Pulmonary delivery of mrna to non-lung target cells

ABSTRACT

Compositions comprising mRNA formulated for pulmonary administration and related methods for delivery of the mRNA and/or encoded protein to a non-lung cell or tissue. The compositions and methods may be used to prevent or ameliorate the symptoms of diseases associated with the mRNA encoded protein.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/657,452, filed on Jun. 8, 2012, the disclosure of which isincorporated herein by reference.

BACKGROUND

Conventional gene therapy involves the use of DNA for insertion ofdesired genetic information into host cells. The DNA introduced into thecell is usually integrated into the genome of one or more transfectedcells, allowing for long-lasting action of the introduced geneticmaterial in the host. While there may be substantial benefits to suchsustained action, integration of exogenous DNA into a host genome mayalso have many deleterious effects. For example, it is possible that theintroduced DNA will be inserted into an intact gene, resulting in amutation which impedes or even totally eliminates the function of theendogenous gene. Thus, gene therapy with DNA may result in theimpairment of a vital genetic function in the treated host, such ase.g., elimination or deleteriously reduced production of an essentialenzyme or interruption of a gene critical for the regulation of cellgrowth, resulting in unregulated or cancerous cell proliferation. Inaddition, with conventional DNA based gene therapy it is necessary foreffective expression of the desired gene product to include a strongpromoter sequence, which again may lead to undesirable changes in theregulation of normal gene expression in the cell. It is also possiblethat the DNA based genetic material will result in the induction ofundesired anti-DNA antibodies, which in turn, may trigger a possiblyfatal immune response.

In contrast to DNA, the use of RNA as a gene therapy agent issubstantially safer because (1) RNA does not involve the risk of beingstably integrated into the genome of the transfected cell, thuseliminating the concern that the introduced genetic material willdisrupt the normal functioning of an essential gene, or cause a mutationthat results in deleterious or oncogenic effects; (2) extraneouspromoter sequences are not required for effective translation of theencoded protein, again avoiding possible deleterious side effects; (3)in contrast to plasmid DNA (pDNA), messenger RNA (mRNA) is devoid ofimmunogenic CpG motifs so that anti-RNA antibodies are not generated;and (4) any deleterious effects that do result from mRNA based on genetherapy would be of limited duration due to the relatively shorthalf-life of RNA. Moreover in many applications, the transient nature ofmRNA transfer to cells, i.e., wherein the duration of any therapeuticeffect is limited by the life span of the mRNA and the protein productin the cells, is more desirable than the potentially longer lastingeffect achieved using DNA based gene therapy. In addition, it is notnecessary for mRNA to enter the nucleus to perform its function, thusavoiding a major barrier to DNA based gene therapy.

One reason that mRNA based gene therapy has not been used more in thepast is that mRNA is far less stable than DNA, especially when itreaches the cytoplasm of a cell and is exposed to degrading enzymes. Thepresence of a hydroxyl group on the second carbon of the sugar moiety inmRNA causes steric hinderance that prevents the mRNA from forming themore stable double helix structure of DNA and thus makes the mRNA moreprone to hydrolytic degradation. As a result, until recently, it waswidely believed that mRNA was too labile to withstand transfectionprotocols.

Advances in RNA stabilizing modifications have sparked more interest inthe use of mRNA in place of plasmid DNA in gene therapy. Yet, in spiteof increased stability of modified mRNA, delivery of mRNA to cells invivo in a manner allowing for therapeutic levels of protein productionis still a challenge, particularly for mRNA encoding full lengthproteins. Some success has been achieved using viral vectors tointroduce mRNA into a host, however mRNA transfection using viralvectors can result in an adverse immune response. In some circumstances,the viral vector may even integrate into the host genome. In addition,production of clinical grade viral vectors is also expensive and timeconsuming. Targeting delivery of the introduced genetic material usingviral vectors can also be difficult to control.

Non-viral delivery of mRNA can be achieved using injection of nakednucleic acids, polyplexes, lipoplexes or liposome entrapped mRNA,biolistic delivery via gene gun, particulate carrier mediated delivery,and electroporation. Non-viral transfection or delivery vehicles aregenerally less-toxic, less immunogenic, and easier and less expensive toprepare than viral vectors for delivery of mRNA. Certain deliveryvehicles, such as cationic lipid or polymer delivery vehicles may alsohelp protect the transfected mRNA from endogenous RNases.

Liposomal delivery of nucleic acids has been employed as a means ofeffectuating the site-specific delivery of encapsulated plasmid DNA,antisense oligonucleotides, short interfering RNA and microRNA-basedtherapies. However the efficient, therapeutically effective, delivery ofmRNAs to targeted cells and tissues, as well as the subsequenttransfection of such targeted cells and tissues remains a technicalchallenge, particularly for delivery of mRNAs encoding full lengthproteins. It is important to design liposomal delivery systems thatprovide sufficient stability to reach desired target cells and theability to efficiently release their encapsulated materials to suchtarget cells to allow for translation of functional protein attherapeutically effective levels.

Many cationic lipids that are employed to construct such liposomal-baseddelivery vehicles are toxic to the targeted cells when used to delivertherapeutically effective amounts of the encapsulated agent.Accordingly, the toxicity associated with cationic lipid represents asignificant obstacle to their general use as non-viral deliveryvehicles, particularly in the quantities necessary to successfullydeliver therapeutically effective amounts of mRNA to target cells.

To date, significant progress using mRNA gene therapy has been made inapplications, particularly for which low levels of translation has notbeen a limiting factor, such as immunization with mRNA encodingantigens. Clinical trials involving vaccination against tumor antigensby intradermal injection of naked or protamine-complexed mRNA havedemonstrated feasibility, lack of toxicity, and promising results. X. Suet al., Mol. Pharmaceutics 8:774-787 (2011). However, low levels oftranslation can restrict the exploitation of mRNA based gene therapy inother applications which require higher levels of sustained stability ofthe mRNA encoded protein to exert a prolonged biological or therapeuticeffect.

In addition, because mRNA gene therapy benefits are relatively transientas compared to DNA based gene therapy, repeated administration, andtypically by injection, are often required to provide long term effects.Thus, more efficient transfection in vivo and the ability to delivermRNA noninvasively and/or to mucosal sites would improve the prospectsfor successful application of mRNA gene therapy.

SUMMARY

The present invention encompasses the surprising discovery thatnanoparticle based formulations of mRNA are able to translocatefollowing pulmonary delivery, i.e., move intact by either active orpassive means from the lung to the systemic blood supply andsubsequently to be deposited in different non-lung cells or tissues,such as, e.g., the liver. This translocation of the nanoparticlecomprising an mRNA encoding a therapeutic protein, such as, e.g.,beta-galactosidase, constitutes non-invasive systemic delivery of anactive pharmaceutical ingredient beyond the lung to result in theproduction of a functional protein to systemically accessible non-lungcells or tissues.

Thus, the present invention provides methods for delivery of mRNA genetherapeutic agents using non-invasive pulmonary administrations. Amongother things, the present invention provides for the delivery of mRNAencoding a protein, which can be used in a method for treating and/orpreventing a disease. In one particular aspect, the invention provides amethod for delivery of messenger RNA (mRNA) to non-lung cell or tissuecomprising administering to the lung a composition comprising mRNAencoding a protein and a lipid carrier vehicle, wherein theadministering to the lung results in the delivery of the mRNA and/or theprotein to a non-lung cell or tissue.

In another aspect, the invention provides a method for delivery of atherapeutic protein to non-lung cell or tissue in a subject comprisingadministering to the lung a composition comprising mRNA encoding atherapeutic protein and a lipid carrier vehicle, such that thetherapeutic protein is delivered to a non-lung cell or tissue.

In another aspect, the invention provides a method of inducing theproduction of a therapeutic protein in a non-lung cell or tissue in asubject comprising administering to the lung a composition comprisingmRNA encoding a therapeutic protein and a lipid carrier vehicle.

In another aspect, the invention provides a method of treating a diseaseor disorder comprising administering to the lung a compositioncomprising mRNA encoding a therapeutic protein and a lipid carriervehicle, wherein the administration to the lung results in the deliveryof the therapeutic protein to a non-lung cell or tissue affected by thedisease or disorder.

In another aspect, the invention provides a composition for pulmonarydelivery of messenger RNA (mRNA) comprising mRNA encoding a protein anda lipid carrier vehicle, wherein the composition is formulated such thatonce administered to the lung, it results in delivery of the mRNA and/orthe protein to a non-lung cell or tissue.

In some embodiments, the composition is administered to the lung byaerosolization. In some embodiments, the composition is delivered to thelung by intratracheal aerosolization. In some embodiments, thecomposition is administered by nebulization. In some embodiments, thecomposition is administered to the lung by instillation. In somespecific embodiments, the composition is administered to the lung of asubject using a device selected form the group consisting of a metereddose inhaler, jet-nebulizer, ultrasonic nebulizer, dry-powder-inhaler,propellant-based inhaler or an insufflator.

In some embodiments, the mRNA comprises a plurality of mRNA species,encoding one or more proteins. In some embodiments, the mRNA comprisesat least two mRNA species, each encoding a different protein. In someembodiments, the mRNA encodes a full length protein. In someembodiments, the mRNA encodes a truncated version of a naturallyoccurring full length protein. In some embodiments, the mRNA encodes oneor more proteins in a single transcript. In some embodiments, the mRNAencodes a chimeric protein, in which one or more protein sequences whichare not naturally associated with the native protein are linked by apeptide bond in the resulting chimeric protein during expression. Insome embodiments, an mRNA suitable for the present invention has alength of or greater than about 0.5 kb, 1 kb, 1.5 kb, 2.0 kb, 2.5 kb,3.0 kb, 3.5 kb, 4.0 kb, 4.5 kb, or 5.0 kb.

In some embodiments, the mRNA encodes an intracellular protein. In someembodiments, the mRNA encodes a cytosolic protein. In some embodiments,the mRNA encodes a protein associated with the actin cytoskeleton. Insome embodiments, the mRNA encodes a protein associated with the plasmamembrane. In some specific embodiments, the mRNA encodes a transmembraneprotein. In some specific embodiments, the mRNA encodes an ion channelprotein. In some embodiments, the mRNA encodes a perinuclear protein. Insome embodiments, the mRNA encodes a nuclear protein. In some specificembodiments, the mRNA encodes a transcription factor. In someembodiments, the mRNA encodes a chaperone protein. In some embodiments,the mRNA encodes an intracellular enzyme. In some embodiments, the mRNAencodes a protein involved in cellular metabolism, DNA repair,transcription and/or translation. In some embodiments, the mRNA encodesan extracellular protein. In some embodiments, the mRNA encodes aprotein associated with the extracellular matrix. In some embodimentsthe mRNA encodes a secreted protein.

In some embodiments, the mRNA encodes a protein (i.e. therapeuticprotein), listed in Table 1, 2, 3 or 4. In some specific embodiments,the protein is selected from the group consisting of alphagalactosidase, erythropoietin, α1-antitrypsin, carboxypeptidase N,alpha-L-iduronidase, iduronate-2-sulfatase,N-acetylglucosamine-1-phosphate transferase, N-acetylglucosaminidase,lysosomal acid lipase, arylsulfatase-A alpha-glucosaminideacetyltransferase, N-acetylglucosamine 6-sulfatase,N-acetylgalactosamine-4-sulfatase, beta-glucosidase, galactose-6-sulfatesulfatase, beta-galactosidase, beta-glucuronidase, glucocerebrosidase,heparan sulfamidase, hyaluronidase, galactocerebrosidase, human growthhormone, ornithine transcarbamylase (OTC), carbamyl phosphatesynthetase-1 (CPS1), argininosuccinate synthetase-1 (ASS1),argininosuccinate lyase (ASL), arginase-1 (ARG1), cystic fibrosistransmembrane conductance regulator (CFTR), Factor VII, Factor VIII,Factor IX, heparan-N-sulfatase, and combinations thereof. In somespecific embodiments, the protein is an intracellular or transmembraneprotein selected from the group consisting of ornithine transcarbamylase(OTC), carbamyl phosphate synthetase-1 (CPS1), argininosuccinatesynthetase-1 (ASS1), argininosuccinate lyase (ASL), arginase-1 (ARG1),cystic fibrosis transmembrane conductance regulator (CFTR), andcombinations thereof.

In some embodiments, the mRNA encodes a protein that is associated witha disease or disorder (i.e., indication) listed in Table 4. In someembodiments, the protein for use in the method, is selected based on itsability to prevent, treat and/or cure a subject affected with a diseaseor disorder (i.e., indication) listed in Table 4. In specificembodiments, the disease or disorder is selected from the groupconsisting of SMN1-related spinal muscular atrophy (SMA), amyotrophiclateral sclerosis (ALS), GALT-related galactosemia, Cystic Fibrosis(CF), SLC3A1-related disorders, cystinuria, COL4A5-related disorders,Alport syndrome, galactocerebrosidase deficiencies, X-linkedadrenoleukodystrophy, adrenomyeloneuropathy, Friedreich's ataxia,Pelizaeus-Merzbacher disease, TSC1 or TSC2-related tuberous sclerosis,Sanfilippo B syndrome (MPS IIIB), CTNS-related cystinosis, theFMR1-related disorders, include Fragile X syndrome, Fragile X-AssociatedTremor/Ataxia Syndrome, Fragile X Premature Ovarian Failure Syndrome,Prader-Willi syndrome, Fabry disease, hereditary hemorrhagictelangiectasia (AT), Niemann-Pick disease Type C1, neuronal ceroidlipofuscinoses-related diseases, Juvenile Neuronal Ceroid Lipofuscinosis(JNCL), Juvenile Batten disease, Santavuori-Haltia disease,Jansky-Bielschowsky disease, PTT-1 deficiency, TPP1 deficiency, EIF2B1,EIF2B2, EIF2B3, EIF2B4 and EIF2B5-related childhood ataxia with centralnervous system hypomyelination/vanishing white matter, CACNA1A andCACNB4-related Episodic Ataxia Type 2, the MECP2-related disorders,Classic Rett Syndrome, MECP2-related Severe Neonatal Encephalopathy,PPM-X Syndrome, CDKL5-related Atypical Rett Syndrome, Kennedy's disease(SBMA), Notch-3 related cerebral autosomal dominant arteriopathy withsubcortical infarcts and leukoencephalopathy (CADASIL), SCN1A andSCN1B-related seizure disorders, Polymerase G-related disorders,Alpers-Huttenlocher syndrome, POLG-related sensory ataxic neuropathy,dysarthria, ophthalmoparesis, autosomal dominant and recessiveprogressive external ophthalmoplegia with mitochondrial DNA deletions,X-Linked adrenal hypoplasia, X-linked agammaglobulinemia, Wilson'sdisease, and blood clotting disorders.

In some embodiments, following delivery to the lung, the mRNA and/orprotein is delivered to a non-lung tissue. In some embodiments, thenon-lung tissue comprises any organ and/or organ system of the body,excluding the lungs. In some specific embodiments, the non-lung tissueis selected from the group consisting of heart, liver, spleen, kidneys,skeletal muscle, lymph nodes, brain skin, cerebrospinal fluid, plasmaand combinations thereof. In some specific embodiments, the non-lungtissue is liver. In some specific embodiments, the non-lung tissue isheart. In some specific embodiments, the non-lung tissue is spleen.

In some embodiments, following delivery to the lung, the mRNA and/orprotein is delivered to a non-lung cell. In some embodiments, thenon-lung cell is selected from the group consisting of hepatocytes,epithelial cells, hematopoietic cells, epithelial cells, endothelialcells, bone cells, stem cells, mesenchymal cells, neural cells, cardiaccells, adipocytes, vascular smooth muscle cells, cardiomyocytes,skeletal muscle cells, beta cells, pituitary cells, synovial liningcells, ovarian cells, testicular cells, fibroblasts, B cells, T cells,reticulocytes, leukocytes, granulocytes, tumor cells, macrophages,neutrophils, antigen presenting cells (dendritic cells), fibroblasts andcombination thereof. In some specific embodiments, the non-lung cell isa hepatocyte.

In some embodiments, the mRNA and/or protein is detectable in thenon-lung cell and/or tissue for at least about 4 hours, 6 hours, 8hours, 10 hours, 12 hours, 18 hours, 24 hours, or more following theadministration to the lung. In some embodiments, the mRNA and/or proteinis detectable in the non-lung cell and/or tissue for at least about 1day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or10 days following the administration to the lung. In some embodiments,the mRNA and/or protein is detectable in the non-lung cell and/or tissuefor at least about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6weeks following administration to the lung. In some embodiments, themRNA is detected using a methods selected the group consisting ofin-situ hybridization, RT-PCR, Real-Time RT-PCR, Northern Blot, nucleaseprotection assay and combinations thereof. In some embodiments, theprotein is detected using a methods selected from the group consistingof Western Blot, ELISA, immunoprecipitation, BCA assay,immunohistochemistry and combinations thereof.

In some embodiments, the mRNA is delivered at an amount greater thanabout 0.5 mg/kg (e.g., greater than about 1.0 mg/kg, 1.5 mg/kg, 2.0mg/kg, 2.5 mg/kg, 3.0 mg/kg, 4.0 mg/kg, 5.0 mg/kg, 6.0 mg/kg, 7.0 mg/kg,8.0 mg/kg, 9.0 mg/kg, or 10.0 mg/kg) body weight of mRNA per dose. Insome embodiments, the mRNA is delivered at an amount ranging from about0.1-100 mg/kg (e.g., about 0.1-90 mg/kg, 0.1-80 mg/kg, 0.1-70 mg/kg,0.1-60 mg/kg, 0.1-50 mg/kg, 0.1-40 mg/kg, 0.1-30 mg/kg, 0.1-20 mg/kg,0.1-10 mg/kg) body weight of mRNA per dose. In some embodiments, themRNA is delivered at an amount of or greater than about 1 mg, 5 mg, 10mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 150 mg, 200mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, or 500 mg per dose.

In some embodiments, the mRNA is encapsulated in a single lipid carriervehicle. In some embodiments, the mRNA is encapsulated in one or morelipid carrier vehicles. In some embodiments, the mRNA is encapsulated inone or more lipid carrier vehicles, which differ in their lipidcomposition, molar ratio of lipid components, size, charge (Zetapotential), targeting ligands and combinations thereof.

In some embodiments, the lipid carrier vehicle is a liposome. In someembodiments, the liposome comprises one or more cationic lipids, one ormore non-cationic lipids, one or more cholesterol-based lipids and oneor more PEG-modified lipids. In some embodiments, the one or morecationic lipid is an ionizable lipid. In some embodiments, the one ormore cationic lipid is a cleavable lipid. In some embodiments, the oneor more cationic lipid is a cholesterol-derived cationic lipid. In someembodiments, the one or more cationic lipids are selected from C12-200,HGT4003, HGT5000, HGT5001, RE-1, RE-2, RE-3, ICE, GL-67, DLinKC2-DMA,DODAP, DODMA, DLinDMA, CLinDMA and combinations thereof.

In some embodiments, the composition further comprises a pulmonarysurfactant. In some embodiments, the composition is formulated asrespirable particles. In some embodiments, the respirable particles havea size less than about 500 μm (e.g., less than about 450 μm, 400 μm, 350μm, 300 μm, 250 μm, 200 μm, 150 μm, 100 μm, or 50 μm). In someembodiments, the composition is formulated as a nebulizable lipid. Insome embodiments, the composition is formulated as a dry powder.

In various embodiments, the invention also provides a compositioncomprising mRNA encoding a protein and a lipid carrier vehicle asdescribed herein for use in a method of delivery of messenger RNA (mRNA)to a non-lung cell or tissue, wherein the method comprises a step ofadministering the composition to the lung of a subject and furtherwherein the administering to the lung results in the delivery of themRNA and/or protein to the non-lung cell or tissue.

In various embodiments, the invention provides a composition comprisingmRNA encoding a protein and a lipid carrier vehicle as described hereinfor use in a method for delivery of therapeutic protein to a non-lungcell or tissue, wherein the method comprises a step of administering thecomposition to the lung of a subject.

In various embodiments, the invention provides a composition comprisingmRNA encoding a protein and a lipid carrier vehicle as described hereinfor use in a method for inducing the production of a protein in anon-lung cell or tissue, wherein the method comprises a step ofadministering the composition to the lung.

In various embodiments, the invention provides a composition comprisingmRNA encoding a protein and a lipid carrier vehicle as described hereinfor use in treating a disease or disorder, wherein the method comprisesa step of administering the composition to the lung and further whereinthe administering to the lung results in the delivery of mRNA and/orprotein to a non-lung cell or tissue affected by the disease ordisorder.

As used in this application, the terms “about” and “approximately” areused as equivalents. Any numerals used in this application with orwithout about/approximately are meant to cover any normal fluctuationsappreciated by one of ordinary skill in the relevant art.

Other features, objects, and advantages of the present invention areapparent in the detailed description that follows. It should beunderstood, however, that the detailed description, while indicatingembodiments of the present invention, is given by way of illustrationonly, not limitation. Various changes and modifications within the scopeof the invention will become apparent to those skilled in the art fromthe detailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows bioluminescence Imaging (BLI) of mice at 6 hrs postintratracheal (IT) spray application. (A) Panels 1, 2, 3 show proteinproduction in mice treated with naked FFL mRNA compared with proteinproduction in mice treated with FFL mRNA inC12-200:DOPE:Cholesterol:DMG-PEG2000 (40:30:25:5) nanoparticles (NPs) inpanels 4, 5, 6. (B) Panels 1, 2 (naked modified FFL mRNA) compared topanels 3, 4, 5 (modified FFL mRNA in C12-200 based NPs).

FIG. 2 shows BLI at 6 hrs post IT spray application using BLI. Panels 1,2, 3 (FFL mRNA in C12-200 based NPs) compared to panels 4, 5, 6(modified FFL mRNA in C12-200 based NPs).

FIG. 3 shows BLI images of mice post IT spray application. (A) All mice(FFL mRNA in C12-200 based NPs); panels 1, 2, 3 after 24 hours comparedto panels 4, 5, 6 after 6 hours. (B) All mice (modified FFL mRNA inC12-200 based NPs); panels 1, 2 after 24 hours compared to panels 3, 4,5 after 6 hours. (C) Panels 1, 2, (FFL mRNA in C12-200 based NPs)compared to panels 3, 4, 5 (modified FFL mRNA in C12-200 based NPs)after 24 hours.

FIG. 4 shows BLI images of mice treated with naked FFL mRNA at 24 hrspost applications. Panels 1, 2 (24 hours after first application);panels 3, 4 (24 hours after second application); panels 5, 6 (24 hoursafter third application).

FIG. 5 shows BLI images of mice treated with naked modified FFL mRNA at24 hrs post application. Panels (24 hours after first application);panels 3, 4 (24 hours after second application); panels 5, 6 (24 hoursafter third application).

FIG. 6 shows BLI images of mice at 6 hours post IT spray application.(A) Panels 1, 2 (FFL mRNA in C12-200 based NPs (10 μg/mouse)); panels 3,4 (FFL mRNA in C12-200 based NPs (5 μg/mouse). (B) Panel 1 (modified FFLmRNA in C12-200 based NPs (10 μg/mouse)); panels 2, 3 (modified FFL mRNAin C12-200 based NPs (5 μg/mouse).

FIG. 7 shows BLI images of mice at 6 hrs and 24 post IT sprayapplication at doses of 10 μg/mouse. (A) All mice (FFL mRNA in C12-200based NPs); panels 1, 2 (24 hours); panels 3, 4 (6 hours). (B) All mice(modified FFL mRNA in C12-200 based NPs); panel 1 (24 hours); panel 2 (6hours). (C) Comparison of panels 1, 2 (FFL mRNA in C12-200 based NPs)with panel 3 (modified FFL mRNA in C12-200 based NPs) at 24 hrs post ITspray.

FIG. 8 shows biodistribution of FFL and modified FFL mRNA in C12-200based NPs at 5 or 10 μg/mouse doses post IT spray.

FIG. 9 shows BLI images of mice at 6 hrs post IT spray application.Panels 1, 2, 3 (modified FFL mRNA in C12-200 based NPs); panels 4, 5(modified FFL mRNA in HGT5001 based NPs)

FIG. 10 shows BLI images of mice at 6 hrs (panels 1, 2) and 24 hrs(panels 3, 4) post IT spray application of modified FFL mRNA inHGT5001:DOPE:Cholesterol:DMG-PEG2000 (40:20:35:5) nanoparticles at dosesof 10 μg/mouse.

FIG. 11 shows BLI image of mice at 24 hrs post IT spray application. (A)Mice with fur removed (modified FFL mRNA in C12-200 based NPs). (B) Micewith fur intact (modified FFL mRNA in C12-200 based NPs).

FIG. 12 shows FFL luminescence detected in lung, liver of mice after asingle, intravenous injection treatment of mRNA-encapsulated lipidnanoparticles formulations, C12-200:DOPE:Cholesterol:DMG-PEG2000(40:30:25:5) and HGT5001:DOPE:Cholesterol:DMG-PEG2000 (40:20:35:5). Micewere sacrificed at 6 hr and 24 hr post-administration.

FIG. 13 shows BLI images of mice 6 hours post nebulization with modifiedFFL mRNA (panels 1, 2) and FFL mRNA (panels 3, 4) in PEI based NPs.

DEFINITIONS

In order for the present invention to be more readily understood,certain terms are first defined below. Additional definitions for thefollowing terms and other terms are set forth throughout thespecification.

Amino acid: As used herein, term “amino acid,” in its broadest sense,refers to any compound and/or substance that can be incorporated into apolypeptide chain. In some embodiments, an amino acid has the generalstructure H₂N—C(H)(R)—COOH. In some embodiments, an amino acid is anaturally occurring amino acid. In some embodiments, an amino acid is asynthetic amino acid; in some embodiments, an amino acid is a d-aminoacid; in some embodiments, an amino acid is an 1-amino acid. “Standardamino acid” refers to any of the twenty standard 1-amino acids commonlyfound in naturally occurring peptides. “Nonstandard amino acid” refersto any amino acid, other than the standard amino acids, regardless ofwhether it is prepared synthetically or obtained from a natural source.As used herein, “synthetic amino acid” encompasses chemically modifiedamino acids, including but not limited to salts, amino acid derivatives(such as amides), and/or substitutions. Amino acids, including carboxy-and/or amino-terminal amino acids in peptides, can be modified bymethylation, amidation, acetylation, protecting groups, and/orsubstitution with other chemical groups that can change the peptide'scirculating half-life without adversely affecting their activity. Aminoacids may participate in a disulfide bond. Amino acids may comprise oneor posttranslational modifications, such as association with one or morechemical entities (e.g., methyl groups, acetate groups, acetyl groups,phosphate groups, formyl moieties, isoprenoid groups, sulfate groups,polyethylene glycol moieties, lipid moieties, carbohydrate moieties,biotin moieties, etc.). The term “amino acid” is used interchangeablywith “amino acid residue,” and may refer to a free amino acid and/or toan amino acid residue of a peptide. It will be apparent from the contextin which the term is used whether it refers to a free amino acid or aresidue of a peptide.

Animal: As used herein, the term “animal” refers to any member of theanimal kingdom. In some embodiments, “animal” refers to humans, at anystage of development. In some embodiments, “animal” refers to non-humananimals, at any stage of development. In certain embodiments, thenon-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit,a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). Insome embodiments, animals include, but are not limited to, mammals,birds, reptiles, amphibians, fish, insects, and/or worms. In someembodiments, an animal may be a transgenic animal,genetically-engineered animal, and/or a clone.

Approximately or about: As used herein, the term “approximately” or“about,” as applied to one or more values of interest, refers to a valuethat is similar to a stated reference value. In certain embodiments, theterm “approximately” or “about” refers to a range of values that fallwithin 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%,8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greaterthan or less than) of the stated reference value unless otherwise statedor otherwise evident from the context (except where such number wouldexceed 100% of a possible value).

Dosing regimen: A “dosing regimen” (or “therapeutic regimen”), as thatterm is used herein, is a set of unit doses (typically more than one)that are administered individually to a subject, typically separated byperiods of time. In some embodiments, a given therapeutic agent has arecommended dosing regiment, which may involve one or more doses. Insome embodiments, a dosing regimen comprises a plurality of doses eachof which are separated from one another by a time period of the samelength; in some embodiments, a dosing regime comprises a plurality ofdoses and at least two different time periods separating individualdoses.

Expression: As used herein, “expression” of a nucleic acid sequencerefers to one or more of the following events: (1) production of an RNAtemplate from a DNA sequence (e.g., by transcription); (2) processing ofan RNA transcript (e.g., by splicing, editing, 5′ cap formation, and/or3′ end formation); (3) translation of an RNA into a polypeptide orprotein; and/or (4) post-translational modification of a polypeptide orprotein. In this application, the terms “expression” and “production,”and grammatical equivalent, are used inter-changeably.

Improve, increase, or reduce: As used herein, the terms “improve,”“increase” or “reduce,” or grammatical equivalents, indicate values thatare relative to a baseline measurement, such as a measurement in thesame individual prior to initiation of the treatment described herein,or a measurement in a control subject (or multiple control subject) inthe absence of the treatment described herein. A “control subject” is asubject afflicted with the same form of disease as the subject beingtreated, who is about the same age as the subject being treated.

In Vitro: As used herein, the term “in vitro” refers to events thatoccur in an artificial environment, e.g., in a test tube or reactionvessel, in cell culture, etc., rather than within a multi-cellularorganism.

In Vivo: As used herein, the term “in vivo” refers to events that occurwithin a multi-cellular organism, such as a human and a non-humananimal. In the context of cell-based systems, the term may be used torefer to events that occur within a living cell (as opposed to, forexample, in vitro systems).

messenger RNA (mRNA): As used herein, the term “messenger RNA (mRNA)”refers to a polynucleotide that encodes at least one polypeptide. mRNAas used herein encompasses both modified and unmodified RNA. mRNA maycontain one or more coding and non-coding regions.

Nucleic acid: As used herein, the term “nucleic acid,” in its broadestsense, refers to any compound and/or substance that is or can beincorporated into a polynucleotide chain. In some embodiments, a nucleicacid is a compound and/or substance that is or can be incorporated intoa polynucleotide chain via a phosphodiester linkage. In someembodiments, “nucleic acid” refers to individual nucleic acid residues(e.g., nucleotides and/or nucleosides). In some embodiments, “nucleicacid” refers to a polynucleotide chain comprising individual nucleicacid residues. In some embodiments, “nucleic acid” encompasses RNA aswell as single and/or double-stranded DNA and/or cDNA. Furthermore, theterms “nucleic acid,” “DNA,” “RNA,” and/or similar terms include nucleicacid analogs, i.e., analogs having other than a phosphodiester backbone.For example, the so-called “peptide nucleic acids,” which are known inthe art and have peptide bonds instead of phosphodiester bonds in thebackbone, are considered within the scope of the present invention. Theterm “nucleotide sequence encoding an amino acid sequence” includes allnucleotide sequences that are degenerate versions of each other and/orencode the same amino acid sequence. Nucleotide sequences that encodeproteins and/or RNA may include introns. Nucleic acids can be purifiedfrom natural sources, produced using recombinant expression systems andoptionally purified, chemically synthesized, etc. Where appropriate,e.g., in the case of chemically synthesized molecules, nucleic acids cancomprise nucleoside analogs such as analogs having chemically modifiedbases or sugars, backbone modifications, etc. A nucleic acid sequence ispresented in the 5′ to 3′ direction unless otherwise indicated. In someembodiments, a nucleic acid is or comprises natural nucleosides (e.g.,adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine,deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs(e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine,3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine,C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine,C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine,8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine);chemically modified bases; biologically modified bases (e.g., methylatedbases); intercalated bases; modified sugars (e.g., 2′-fluororibose,ribose, 2′-deoxyribose, arabinose, and hexose); and/or modifiedphosphate groups (e.g., phosphorothioates and 5′-N-phosphoramiditelinkages). In some embodiments, the present invention is specificallydirected to “unmodified nucleic acids,” meaning nucleic acids (e.g.,polynucleotides and residues, including nucleotides and/or nucleosides)that have not been chemically modified in order to facilitate or achievedelivery.

Patient: As used herein, the term “patient” or “subject” refers to anyorganism to which a provided composition may be administered, e.g., forexperimental, diagnostic, prophylactic, cosmetic, and/or therapeuticpurposes. Typical patients include animals (e.g., mammals such as mice,rats, rabbits, non-human primates, and/or humans). In some embodiments,a patient is a human. A human includes pre and post natal forms.

Pharmaceutically acceptable: The term “pharmaceutically acceptable” asused herein, refers to substances that, within the scope of soundmedical judgment, are suitable for use in contact with the tissues ofhuman beings and animals without excessive toxicity, irritation,allergic response, or other problem or complication, commensurate with areasonable benefit/risk ratio.

Polypeptide: As used herein, a “polypeptide”, generally speaking, is astring of at least two amino acids attached to one another by a peptidebond. In some embodiments, a polypeptide may include at least 3-5 aminoacids, each of which is attached to others by way of at least onepeptide bond. Those of ordinary skill in the art will appreciate thatpolypeptides sometimes include “non-natural” amino acids or otherentities that nonetheless are capable of integrating into a polypeptidechain, optionally.

Protein: As used herein, the term “protein” of “therapeutic protein”refers to a polypeptide (i.e., a string of at least two amino acidslinked to one another by peptide bonds). Proteins may include moietiesother than amino acids (e.g., may be glycoproteins, proteoglycans, etc.)and/or may be otherwise processed or modified. Those of ordinary skillin the art will appreciate that a “protein” can be a completepolypeptide chain as produced by a cell (with or without a signalsequence), or can be a characteristic portion thereof. Those of ordinaryskill will appreciate that a protein can sometimes include more than onepolypeptide chain, for example linked by one or more disulfide bonds orassociated by other means. Polypeptides may contain 1-amino acids,d-amino acids, or both and may contain any of a variety of amino acidmodifications or analogs known in the art. Useful modifications include,e.g., terminal acetylation, amidation, methylation, etc. In someembodiments, proteins may comprise natural amino acids, non-naturalamino acids, synthetic amino acids, and combinations thereof. The term“peptide” is generally used to refer to a polypeptide having a length ofless than about 100 amino acids, less than about 50 amino acids, lessthan 20 amino acids, or less than 10 amino acids. In some embodiments,proteins are antibodies, antibody fragments, biologically activeportions thereof, and/or characteristic portions thereof.

Subject: As used herein, the term “subject” refers to a human or anynon-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine,sheep, horse or primate). A human includes pre- and post-natal forms. Inmany embodiments, a subject is a human being. A subject can be apatient, which refers to a human presenting to a medical provider fordiagnosis or treatment of a disease. The term “subject” is used hereininterchangeably with “individual” or “patient.” A subject can beafflicted with or is susceptible to a disease or disorder but may or maynot display symptoms of the disease or disorder.

Therapeutically effective amount: As used herein, the term“therapeutically effective amount” of a therapeutic agent means anamount that is sufficient, when administered to a subject suffering fromor susceptible to a disease, disorder, and/or condition, to treat,diagnose, prevent, and/or delay the onset of the symptom(s) of thedisease, disorder, and/or condition. It will be appreciated by those ofordinary skill in the art that a therapeutically effective amount istypically administered via a dosing regimen comprising at least one unitdose.

Treatment: As used herein, the term “treatment” (also “treat” or“treating”) refers to any administration of a substance (e.g., providedcompositions) that partially or completely alleviates, ameliorates,relives, inhibits, delays onset of, reduces severity of, and/or reducesincidence of one or more symptoms, features, and/or causes of aparticular disease, disorder, and/or condition (e.g., influenza). Suchtreatment may be of a subject who does not exhibit signs of the relevantdisease, disorder and/or condition and/or of a subject who exhibits onlyearly signs of the disease, disorder, and/or condition. Alternatively oradditionally, such treatment may be of a subject who exhibits one ormore established signs of the relevant disease, disorder and/orcondition. In some embodiments, treatment may be of a subject who hasbeen diagnosed as suffering from the relevant disease, disorder, and/orcondition. In some embodiments, treatment may be of a subject known tohave one or more susceptibility factors that are statisticallycorrelated with increased risk of development of the relevant disease,disorder, and/or condition.

DETAILED DESCRIPTION

The present invention provides, among other things, methods andcompositions for systemic delivery of mRNA and/or its protein productbased on pulmonary delivery. In some embodiments, the present inventionprovides a method of administering a composition comprising mRNA and alipid carrier vehicle to the lungs of a subject, for delivery of themRNA and/or protein to non-lung cells and tissues. In some embodiments,mRNA encoding a single protein are delivered. In some embodiments one ormore mRNA species encoding one or more proteins are delivered. In someembodiments, the mRNA is delivered using a single lipid carrier vehicle(e.g. liposome or lipid-derived nanoparticle). In some embodiments themRNA is delivered using a one or more lipid carrier vehicles.

Various aspects of the invention are described in detail in thefollowing sections. The use of sections is not meant to limit theinvention. Each section can apply to any aspect of the invention. Inthis application, the use of “or” means “and/or” unless statedotherwise.

mRNA and mRNA Synthesis

mRNAs according to the present invention may be synthesized according toany of a variety of known methods. For example, mRNAs according to thepresent invention may be synthesized via in vitro transcription (IVT).Briefly, IVT is typically performed with a linear or circular DNAtemplate containing a promoter, a pool of ribonucleotide triphosphates,a buffer system that may include DTT and magnesium ions, and anappropriate RNA polymerase (e.g., T3, T7 or SP6 RNA polymerase), DNAseI, pyrophosphatase, and/or RNAse inhibitor. The exact conditions willvary according to the specific application.

In some embodiments, for the preparation of mRNA according to theinvention, a DNA template is transcribed in vitro. A suitable DNAtemplate typically has a promoter, for example a T3, T7 or SP6 promoter,for in vitro transcription, followed by desired nucleotide sequence fora desired mRNA and a termination signal.

Desired mRNA sequence according to the invention may be determined andincorporated into a DNA template using standard methods. For example,starting from a desired amino acid sequence a virtual reversetranslation is carried out based on the degenerated genetic code.Optimization algorithms may then be used for selection of suitablecodons. Typically, the G/C content can be optimized to achieve thehighest possible G/C content on one hand, taking into the best possibleaccount the frequency of the tRNAs according to codon usage on the otherhand. The optimized RNA sequence can be established and displayed, forexample, with the aid of an appropriate display device and compared withthe original (wild-type) sequence. A secondary structure can also beanalyzed to calculate stabilizing and destabilizing properties or,respectively, regions of the RNA.

mRNA according to the present invention may be synthesized as unmodifiedor modified mRNA. In some embodiments, the mRNA may include one or morechemical or structural modifications to abrogate mRNA interaction withtoll-like receptors TLR3, TLR7, TLR8, and retinoid-inducible gene I(RIG-I) to reduce immunogenicity as well as improve stability of themRNA.

For example, in certain embodiments, the mRNA may be modified asdescribed in U.S. patent publication 2009/0286852 (incorporated hereinby reference), to comprise one or more pseudouridine residues. Kormannet al., Nature Biotechnology 29(2):154-157 (2011) describe replacementof uridine and cytidine with 2-thiouridine and 5-methylcytidine tosynergistically decrease mRNA binding to pattern recognition receptorsTLR3, TLR7, TLR8, and RIG-I and increase stability of the mRNA. SeeEP2459231. In yet other embodiments, the mRNA may be modified to reduceimmunogenicity as described in European Application EP10742089(incorporated herein by reference).

In other embodiments, modifications of mRNA can include, for example,modifications of the nucleotides of the RNA. An modified mRNA accordingto the invention can thus include, for example, backbone modifications,sugar modifications or base modifications. In some embodiments, mRNAsencoding a protein of interest may be synthesized from naturallyoccurring nucleotides and/or nucleotide analogues (modified nucleotides)including, but not limited to, purines (adenine (A), guanine (G)) orpyrimidines (thymine (T), cytosine (C), uracil (U)), and as modifiednucleotides analogues or derivatives of purines and pyrimidines, such ase.g. 1-methyl-adenine, 2-methyl-adenine,2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine,N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine,4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine,1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine,7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5-uracil),dihydro-uracil, 2-thio-uracil, 4-thio-uracil,5-carboxymethylaminomethyl-2-thio-uracil,5-(carboxyhydroxymethyl)-uracil, 5-fluoro-uracil, 5-bromo-uracil,5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil,5-methyl-uracil, N-uracil-5-oxyacetic acid methyl ester,5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil,5′-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyaceticacid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil,queosine, .beta.-D-mannosyl-queosine, wybutoxosine, andphosphoramidates, phosphorothioates, peptide nucleotides,methylphosphonates, 7-deazaguanosine, 5-methylcytosine and inosine. Thepreparation of such analogues is known to a person skilled in the arte.g. from the U.S. Pat. No. 4,373,071, U.S. Pat. No. 4,401,796, U.S.Pat. No. 4,415,732, U.S. Pat. No. 4,458,066, U.S. Pat. No. 4,500,707,U.S. Pat. No. 4,668,777, U.S. Pat. No. 4,973,679, U.S. Pat. No.5,047,524, U.S. Pat. No. 5,132,418, U.S. Pat. No. 5,153,319, U.S. Pat.Nos. 5,262,530 and 5,700,642, the disclosure of which is included herein its full scope by reference. See G. Tavernier et al., J. ControlledRelease 150:238-247 (2011) and WO 2010/053572, incorporated herein byreference. See also US 2009/0286852 providing an extensive list ofmodified nucleosides, at ¶¶55, and 68-75 and WO 2008/052770(incorporated herein by reference) describing numerous mRNAmodifications for increasing mRNA stability and protein production.

In some embodiments, mRNAs may contain RNA backbone modifications.Typically, a backbone modification is a modification in which thephosphates of the backbone of the nucleotides contained in the RNA aremodified chemically. Exemplary backbone modifications typically include,but are not limited to, modifications from the group consisting ofmethylphosphonates, methylphosphoramidates, phosphoramidates,phosphorothioates (e.g. cytidine 5′-O-(1-thiophosphate)),boranophosphates, positively charged guanidinium groups etc., whichmeans by replacing the phosphodiester linkage by other anionic, cationicor neutral groups.

In some embodiments, mRNAs may contain sugar modifications. A typicalsugar modification is a chemical modification of the sugar of thenucleotides it contains including, but not limited to, sugarmodifications chosen from the group consisting of2′-deoxy-2′-fluoro-oligoribonucleotide (2′-fluoro-2′-deoxycytidine5′-triphosphate, 2′-fluoro-2′-deoxyuridine 5′-triphosphate),2′-deoxy-2′-deamine-oligoribonucleotide (2′-amino-2′-deoxycytidine5′-triphosphate, 2′-amino-2′-deoxyuridine 5′-triphosphate),2′-O-alkyloligoribonucleotide, 2′-deoxy-2′-C-alkyloligoribonucleotide(2′-O-methylcytidine 5′-triphosphate, 2′-methyluridine 5′-triphosphate),2′-C-alkyloligoribonucleotide, and isomers thereof (2′-aracytidine5′-triphosphate, 2′-arauridine 5′-triphosphate), or azidotriphosphates(2′-azido-2′-deoxycytidine 5′-triphosphate, 2′-azido-2′-deoxyuridine5′-triphosphate).

In some embodiments, mRNAs may contain modifications of the bases of thenucleotides (base modifications). A modified nucleotide which contains abase modification is also called a base-modified nucleotide. Examples ofsuch base-modified nucleotides include, but are not limited to,2-amino-6-chloropurine riboside 5′-triphosphate, 2-aminoadenosine5′-triphosphate, 2-thiocytidine 5′-triphosphate, 2-thiouridine5′-triphosphate, 4-thiouridine 5′-triphosphate, 5-aminoallylcytidine5′-triphosphate, 5-aminoallyluridine 5′-triphosphate, 5-bromocytidine5′-triphosphate, 5-bromouridine 5′-triphosphate, 5-iodocytidine5′-triphosphate, 5-iodouridine 5′-triphosphate, 5-methylcytidine5′-triphosphate, 5-methyluridine 5′-triphosphate, 6-azacytidine5′-triphosphate, 6-azauridine 5′-triphosphate, 6-chloropurine riboside5′-triphosphate, 7-deazaadenosine 5′-triphosphate, 7-deazaguanosine5′-triphosphate, 8-azaadenosine 5′-triphosphate, 8-azidoadenosine5′-triphosphate, benzimidazole riboside 5′-triphosphate,N1-methyladenosine 5′-triphosphate, N1-methylguanosine 5′-triphosphate,N6-methyladenosine 5′-triphosphate, 06-methylguanosine 5′-triphosphate,pseudouridine 5′-triphosphate, puromycin 5′-triphosphate or xanthosine5′-triphosphate.

In certain embodiments, stabilizing modifications may be made to eitheror both the 3′ and 5′ ends of the mRNA and include, e.g., end capping,polyA tail, replacement of unstable non-coding sequences (such asadenylate uridylate rich elements (AREs) or addition or 3′ or 5′untranslated sequences from stable mRNA (such as, e.g., β-globin, actin,GAPDH, tubulin, histone, or citric acid cycle enzyme mRNA). Stabilizingmodifications may also be made within the mRNA, and include, e.g., codonoptimization and/or modification of the Kozak sequence. and/orincorporation of modified nucleosides (such as, e.g.,pyrrolo-pyrimidine, C5-iodouridine, 2-amino adenosine, and2-thiothymidine). In certain embodiments, the modified mRNA used in themethods and compositions of the invention include a 5′ untranslatedsequence from CMV immediate-early 1 (IE1) gene:XCAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACG, wherein X, if present is GGA (SEQ IDNO:1), or a sequence that is at least 90% or at least 95% identical toSEQ ID NO:1, or a and/or a 3′untranslated sequence from human growthhormone (hGH) gene:CGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUC (SEQ ID NO:2), or asequence that is at least 90% or at least 95% identical to SEQ ID NO:2,to improve the nuclease resistance and/or improve the half-life of themRNA. In addition to increasing the stability of the mRNA polynucleotidesequence, it has been surprisingly discovered the inclusion of theuntranslated sequence of CMV immediate-early 1 (IE1) gene and/or theuntranslated sequence from the hGH gene further enhances the translationof the mRNA.

Typically, mRNA synthesis includes the addition of a “cap” on theN-terminal (5′) end, and a “tail” on the C-terminal (3′) end. Thepresence of the cap is important in providing resistance to nucleasesfound in most eukaryotic cells. The presence of a “tail” serves toprotect the mRNA from exonuclease degradation.

Thus, in some embodiments, mRNAs of the current invention include a 5′cap structure. A 5′ cap is typically added as follows: first, an RNAterminal phosphatase removes one of the terminal phosphate groups fromthe 5′ nucleotide, leaving two terminal phosphates; guanosinetriphosphate (GTP) is then added to the terminal phosphates via aguanylyl transferase, producing a 5′5′5 triphosphate linkage; and the7-nitrogen of guanine is then methylated by a methyltransferase.Examples of cap structures include, but are not limited to, m7G(5′)ppp(5′(A,G(5′)ppp(5′)A and G(5′)ppp(5′)G.

In some embodiments, mRNAs of the current invention include a 3′ poly(A)tail structure. A poly-A tail on the 3′ terminus of mRNA typicallyincludes about 10 to 300 adenosine nucleotides (e.g., about 10 to 200adenosine nucleotides, about 10 to 150 adenosine nucleotides, about 10to 100 adenosine nucleotides, about 20 to 70 adenosine nucleotides, orabout 20 to 60 adenosine nucleotides). In some embodiments, mRNAs of thecurrent invention include a 3′ poly(C) tail structure. A suitable poly-Ctail on the 3′ terminus of mRNA typically include about 10 to 200cytosine nucleotides (e.g., about 10 to 150 cytosine nucleotides, about10 to 100 cytosine nucleotides, about 20 to 70 cytosine nucleotides,about 20 to 60 cytosine nucleotides, or about 10 to 40 cytosinenucleotides). The poly-C tail may be added to the poly-A tail or maysubstitute the poly-A tail.

In some embodiments, mRNAs of the current invention include a 5′ and/or3′ untranslated region. In some embodiments, a 5′ untranslated regionincludes one or more elements that affect an mRNA's stability ortranslation, for example, an iron responsive element. In someembodiments, a 5′ untranslated region may be between about 50 and 500nucleotides in length.

In some embodiments, a 3′ untranslated region includes one or more of apolyadenylation signal, a binding site for proteins that affect anmRNA's stability of location in a cell, or one or more binding sites formiRNAs. In some embodiments, a 3′ untranslated region may be between 50and 500 nucleotides in length or longer.

Proteins Encoded by mRNAs

The mRNAs used in the compositions and methods of the invention may beused to express full length, truncated, native or modified protein fordelivery to non-lung tissue and cells. In some embodiments, the mRNAcomprises at least one mRNA species encoding a protein (i.e. atherapeutic protein). In some embodiments, the mRNA comprises aplurality of mRNA species, encoding one or more gene products. In someembodiments, the mRNA comprises at least two mRNA species, each encodinga different gene product. In some embodiments, the mRNA encodes a fulllength protein. In some embodiments, the mRNA encode a truncated versionof the naturally occurring full length protein. In some embodiments, themRNA encode one or more truncated protein from different gene productsin a single transcript. In some embodiments, the mRNA encodes a chimericprotein, in which one or more protein sequences which are not naturallyassociated with the native protein are linked by a peptide bond in theresulting chimeric protein during expression. In some embodiments, themRNA may be used to express a partial or full length protein comprisingcellular activity at a level equal to or greater than that of the nativeprotein. In some embodiments, the mRNA may be used to express a partialor full length protein with cellular activity at a level equal to orless than that of the native protein.

In some embodiments the mRNA encodes an intracellular protein. In someembodiments, the mRNA encodes a cytosolic protein. In some embodiments,the mRNA encodes a protein associated with the actin cytoskeleton. Insome embodiments, the mRNA encodes a protein associated with the plasmamembrane. In some specific embodiments, the mRNA encodes a transmembraneprotein. In some specific embodiments the mRNA encodes an ion channelprotein. In some embodiments, the mRNA encodes a perinuclear protein. Insome embodiments, the mRNA encodes a nuclear protein. In some specificembodiments, the mRNA encodes a transcription factor. In someembodiments, the mRNA encodes a chaperone protein. In some embodiments,the mRNA encodes an intracellular enzyme (e.g., mRNA encoding an enzymeassociated with urea cycle or lysosomal storage metabolic disorders). Insome embodiments, the mRNA encodes a protein involved in cellularmetabolism, DNA repair, transcription and/or translation. In someembodiments, the mRNA encodes an extracellular protein. In someembodiments, the mRNA encodes a protein associated with theextracellular matrix. In some embodiments the mRNA encodes a secretedprotein. In specific embodiments, the mRNA used in the composition andmethods of the invention may be used to express functional proteins orenzymes that are excreted or secreted by one or more target cells intothe surrounding extracellular fluid (e.g., mRNA encoding hormones andneurotransmitters)

In some embodiments, the compositions and methods of the inventionprovide for delivery of mRNA encoding a secreted protein. In someembodiments, the compositions and methods of the invention provide fordelivery of mRNA encoding one or more secreted proteins listed in Table1; thus, compositions of the invention may comprise an mRNA encoding aprotein listed in Table 1 (or a homolog thereof, as discussed below)along with other components set out herein, and methods of the inventionmay comprise preparing and/or administering a composition comprising anmRNA encoding a protein listed in Table 1 (or a homolog thereof, asdiscussed below) along with other components set out herein.

TABLE 1 Secreted Proteins Uniprot ID Protein Name Gene Name A1E959Odontogenic ameloblast-associated protein ODAM A1KZ92 Peroxidasin-likeprotein PXDNL A1L453 Serine protease 38 PRSS38 A1L4H1 Soluble scavengerreceptor cysteine-rich domain- SSC5D containing protein SSC5D A2RUU4Colipase-like protein 1 CLPSL1 A2VDF0 Fucose mutarotase FUOM A2VEC9SCO-spondin SSPO A3KMH1 von Willebrand factor A domain-containing VWA8protein 8 A4D0S4 Laminin subunit beta-4 LAMB4 A4D1T9 Probable inactiveserine protease 37 PRSS37 A5D8T8 C-type lectin domain family 18 member ACLEC18A A6NC86 phospholipase A2 inhibitor and Ly6/PLAUR PINLYPdomain-containing protein A6NCI4 von Willebrand factor Adomain-containing VWA3A protein 3A A6ND01 Probable folate receptor deltaFOLR4 A6NDD2 Beta-defensin 108B-like A6NE02 BTB/POZ domain-containingprotein 17 BTBD17 A6NEF6 Growth hormone 1 GH1 A6NF02 NPIP-like proteinLOC730153 A6NFB4 HCG1749481, isoform CRA_k CSH1 A6NFZ4 Protein FAM24AFAM24A A6NG13 Glycosyltransferase 54 domain-containing protein A6NGN9IgLON family member 5 IGLON5 A6NHN0 Otolin-1 OTOL1 A6NHN6 Nuclear porecomplex-interacting protein-like 2 NPIPL2 A6NI73 Leukocyteimmunoglobulin-like receptor LILRA5 subfamily A member 5 A6NIT4Chorionic somatomammotropin hormone 2 CSH2 isoform 2 A6NJ69 IgA-inducingprotein homolog IGIP A6NKQ9 Choriogonadotropin subunit beta variant 1CGB1 A6NMZ7 Collagen alpha-6(VI) chain COL6A6 A6NNS2Dehydrogenase/reductase SDR family member 7C DHRS7C A6XGL2 Insulin Achain INS A8K0G1 Protein Wnt WNT7B A8K2U0 Alpha-2-macroglobulin-likeprotein 1 A2ML1 A8K7I4 Calcium-activated chloride channel regulator 1CLCA1 A8MTL9 Serpin-like protein HMSD HMSD A8MV23 Serpin E3 SERPINE3A8MZH6 Oocyte-secreted protein 1 homolog OOSP1 A8TX70 Collagenalpha-5(VI) chain COL6A5 B0ZBE8 Natriuretic peptide NPPA B1A4G9Somatotropin GH1 B1A4H2 HCG1749481, isoform CRA_d CSH1 B1A4H9 Chorionicsomatomammotropin hormone CSH2 B1AJZ6 Protein Wnt WNT4 B1AKI9 Isthmin-1ISM1 B2RNN3 Complement C1q and tumor necrosis factor- C1QTNF9B relatedprotein 9B B2RUY7 von Willebrand factor C domain-containing VWC2Lprotein 2-like B3GLJ2 Prostate and testis expressed protein 3 PATE3B4DI03 SEC11-like 3 (S. cerevisiae), isoform CRA_a SEC11L3 B4DJF9Protein Wnt WNT4 B4DUL4 SEC11-like 1 (S. cerevisiae), isoform CRA_dSEC11L1 B5MCC8 Protein Wnt WNT10B B8A595 Protein Wnt WNT7B B8A597Protein Wnt WNT7B B8A598 Protein Wnt WNT7B B9A064 Immunoglobulinlambda-like polypeptide 5 IGLL5 C9J3H3 Protein Wnt WNT10B C9J8I8 ProteinWnt WNT5A C9JAF2 Insulin-like growth factor II Ala-25 Del IGF2 C9JCI2Protein Wnt WNT10B C9JL84 HERV-H LTR-associating protein 1 HHLA1 C9JNR5Insulin A chain INS C9JUI2 Protein Wnt WNT2 D6RF47 Protein Wnt WNT8AD6RF94 Protein Wnt WNT8A E2RYF7 Protein PBMUCL2 HCG22 E5RFR1PENK(114-133) PENK E7EML9 Serine protease 44 PRSS44 E7EPC3 Protein WntWNT9B E7EVP0 Nociceptin PNOC E9PD02 Insulin-like growth factor I IGF1E9PH60 Protein Wnt WNT16 E9PJL6 Protein Wnt WNT11 F5GYM2 Protein WntWNT5B F5H034 Protein Wnt WNT5B F5H364 Protein Wnt WNT5B F5H7Q6 ProteinWnt WNT5B F8WCM5 Protein INS-IGF2 INS-IGF2 F8WDR1 Protein Wnt WNT2H0Y663 Protein Wnt WNT4 H0YK72 Signal peptidase complex catalyticsubunit SEC11A SEC11A H0YK83 Signal peptidase complex catalytic subunitSEC11A SEC11A H0YM39 Chorionic somatomammotropin hormone CSH2 H0YMT7Chorionic somatomammotropin hormone CSH1 H0YN17 Chorionicsomatomammotropin hormone CSH2 H0YNA5 Signal peptidase complex catalyticsubunit SEC11A SEC11A H0YNG3 Signal peptidase complex catalytic subunitSEC11A SEC11A H0YNX5 Signal peptidase complex catalytic subunit SEC11ASEC11A H7BZB8 Protein Wnt WNT10A H9KV56 Choriogonadotropin subunit betavariant 2 CGB2 I3L0L8 Protein Wnt WNT9B J3KNZ1 Choriogonadotropinsubunit beta variant 1 CGB1 J3KP00 Choriogonadotropin subunit beta CGB7J3QT02 Choriogonadotropin subunit beta variant 1 CGB1 O00175 C-C motifchemokine 24 CCL24 O00182 Galectin-9 LGALS9 O00187 Mannan-binding lectinserine protease 2 MASP2 O00230 Cortistatin CORT O00253 Agouti-relatedprotein AGRP O00270 12-(S)-hydroxy-5,8,10,14-eicosatetraenoic acid GPR31receptor O00292 Left-right determination factor 2 LEFTY2 O00294Tubby-related protein 1 TULP1 O00295 Tubby-related protein 2 TULP2O00300 Tumor necrosis factor receptor superfamily TNFRSF11B member 11BO00339 Matrilin-2 MATN2 O00391 Sulfhydryl oxidase 1 QSOX1 O00468 AgrinAGRN O00515 Ladinin-1 LAD1 O00533 Processed neural cell adhesionmolecule L1-like CHL1 protein O00584 Ribonuclease T2 RNASET2 O00585 C-Cmotif chemokine 21 CCL21 O00602 Ficolin-1 FCN1 O00622 Protein CYR61CYR61 O00626 MDC(5-69) CCL22 O00634 Netrin-3 NTN3 O00744 Protein Wnt-10bWNT10B O00755 Protein Wnt-7a WNT7A O14498 Immunoglobulin superfamilycontaining leucine- ISLR rich repeat protein O14511 Pro-neuregulin-2,membrane-bound isoform NRG2 O14594 Neurocan core protein NCAN O14625C-X-C motif chemokine 11 CXCL11 O14638 Ectonucleotide ENPP3pyrophosphatase/phosphodiesterase family member 3 O14656 Torsin-1A TOR1AO14657 Torsin-1B TOR1B O14786 Neuropilin-1 NRP1 O14788 Tumor necrosisfactor ligand superfamily member TNFSF11 11, membrane form O14791Apolipoprotein L1 APOL1 O14793 Growth/differentiation factor 8 MSTNO14904 Protein Wnt-9a WNT9A O14905 Protein Wnt-9b WNT9B O14944Proepiregulin EREG O14960 Leukocyte cell-derived chemotaxin-2 LECT2O15018 Processed PDZ domain-containing protein 2 PDZD2 O15041Semaphorin-3E SEMA3E O15072 A disintegrin and metalloproteinase withADAMTS3 thrombospondin motifs 3 O15123 Angiopoietin-2 ANGPT2 O15130Neuropeptide FF NPFF O15197 Ephrin type-B receptor 6 EPHB6 O15204 ADAMDEC1 ADAMDEC1 O15230 Laminin subunit alpha-5 LAMA5 O15232 Matrilin-3MATN3 O15240 Neuroendocrine regulatory peptide-1 VGF O15263Beta-defensin 4A DEFB4A O15335 Chondroadherin CHAD O15393 Transmembraneprotease serine 2 catalytic chain TMPRSS2 O15444 C-C motif chemokine 25CCL25 O15467 C-C motif chemokine 16 CCL16 O15496 Group 10 secretoryphospholipase A2 PLA2G10 O15520 Fibroblast growth factor 10 FGF10 O15537Retinoschisin RS1 O43157 Plexin-B1 PLXNB1 O43184 Disintegrin andmetalloproteinase domain- ADAM12 containing protein 12 O43240Kallikrein-10 KLK10 O43278 Kunitz-type protease inhibitor 1 SPINT1O43320 Fibroblast growth factor 16 FGF16 O43323 Desert hedgehog proteinC-product DHH O43405 Cochlin COCH O43508 Tumor necrosis factor ligandsuperfamily member TNFSF12 12, membrane form O43555 Progonadoliberin-2GNRH2 O43557 Tumor necrosis factor ligand superfamily member TNFSF14 14,soluble form O43692 Peptidase inhibitor 15 PI15 O43699 Sialicacid-binding Ig-like lectin 6 SIGLEC6 O43820 Hyaluronidase-3 HYAL3O43827 Angiopoietin-related protein 7 ANGPTL7 O43852 Calumenin CALUO43854 EGF-like repeat and discoidin I-like domain- EDIL3 containingprotein 3 O43866 CD5 antigen-like CD5L O43897 Tolloid-like protein 1TLL1 O43915 Vascular endothelial growth factor D FIGF O43927 C-X-C motifchemokine 13 CXCL13 O60218 Aldo-keto reductase family 1 member B10AKR1B10 O60235 Transmembrane protease serine 11D TMPRSS11D O60258Fibroblast growth factor 17 FGF17 O60259 Kallikrein-8 KLK8 O60383Growth/differentiation factor 9 GDF9 O60469 Down syndrome cell adhesionmolecule DSCAM O60542 Persephin PSPN O60565 Gremlin-1 GREM1 O60575Serine protease inhibitor Kazal-type 4 SPINK4 O60676 Cystatin-8 CST8O60687 Sushi repeat-containing protein SRPX2 SRPX2 O60844 Zymogengranule membrane protein 16 ZG16 O60882 Matrix metalloproteinase-20MMP20 O60938 Keratocan KERA O75015 Low affinity immunoglobulin gamma Fcregion FCGR3B receptor III-B O75077 Disintegrin and metalloproteinasedomain- ADAM23 containing protein 23 O75093 Slit homolog 1 protein SLIT1O75094 Slit homolog 3 protein SLIT3 O75095 Multiple epidermal growthfactor-like domains MEGF6 protein 6 O75173 A disintegrin andmetalloproteinase with ADAMTS4 thrombospondin motifs 4 O75200 Nuclearpore complex-interacting protein-like 1 NPIPL1 O75339 Cartilageintermediate layer protein 1 C1 CILP O75354 Ectonucleoside triphosphatediphosphohydrolase 6 ENTPD6 O75386 Tubby-related protein 3 TULP3 O75398Deformed epidermal autoregulatory factor 1 DEAF1 homolog O75443Alpha-tectorin TECTA O75445 Usherin USH2A O75462 Cytokine receptor-likefactor 1 CRLF1 O75487 Glypican-4 GPC4 O75493 Carbonic anhydrase-relatedprotein 11 CA11 O75594 Peptidoglycan recognition protein 1 PGLYRP1O75596 C-type lectin domain family 3 member A CLEC3A O75610 Left-rightdetermination factor 1 LEFTY1 O75629 Protein CREG1 CREG1 O75636Ficolin-3 FCN3 O75711 Scrapie-responsive protein 1 SCRG1 O75715Epididymal secretory glutathione peroxidase GPX5 O75718Cartilage-associated protein CRTAP O75829 Chondrosurfactant proteinLECT1 O75830 Serpin I2 SERPINI2 O75882 Attractin ATRN O75888 Tumornecrosis factor ligand superfamily member TNFSF13 13 O75900 Matrixmetalloproteinase-23 MMP23A O75951 Lysozyme-like protein 6 LYZL6 O75973C1q-related factor C1QL1 O76038 Secretagogin SCGN O76061 Stanniocalcin-2STC2 O76076 WNT1-inducible-signaling pathway protein 2 WISP2 O76093Fibroblast growth factor 18 FGF18 O76096 Cystatin-F CST7 O94769Extracellular matrix protein 2 ECM2 O94813 Slit homolog 2 proteinC-product SLIT2 O94907 Dickkopf-related protein 1 DKK1 O94919Endonuclease domain-containing 1 protein ENDOD1 O94964 N-terminal formSOGA1 O95025 Semaphorin-3D SEMA3D O95084 Serine protease 23 PRSS23O95150 Tumor necrosis factor ligand superfamily member TNFSF15 15 O95156Neurexophilin-2 NXPH2 O95157 Neurexophilin-3 NXPH3 O95158Neurexophilin-4 NXPH4 O95388 WNT1-inducible-signaling pathway protein 1WISP1 O95389 WNT1-inducible-signaling pathway protein 3 WISP3 O95390Growth/differentiation factor 11 GDF11 O95393 Bone morphogenetic protein10 BMP10 O95399 Urotensin-2 UTS2 O95407 Tumor necrosis factor receptorsuperfamily TNFRSF6B member 6B O95428 Papilin PAPLN O95445Apolipoprotein M APOM O95450 A disintegrin and metalloproteinase withADAMTS2 thrombospondin motifs 2 O95460 Matrilin-4 MATN4 O95467 LHALtetrapeptide GNAS O95631 Netrin-1 NTN1 O95633 Follistatin-relatedprotein 3 FSTL3 O95711 Lymphocyte antigen 86 LY86 O95715 C-X-C motifchemokine 14 CXCL14 O95750 Fibroblast growth factor 19 FGF19 O95760Interleukin-33 IL33 O95813 Cerberus CER1 O95841 Angiopoietin-relatedprotein 1 ANGPTL1 O95897 Noelin-2 OLFM2 O95925 Eppin EPPIN O95965Integrin beta-like protein 1 ITGBL1 O95967 EGF-containing fibulin-likeextracellular matrix EFEMP2 protein 2 O95968 Secretoglobin family 1Dmember 1 SCGB1D1 O95969 Secretoglobin family 1D member 2 SCGB1D2 O95970Leucine-rich glioma-inactivated protein 1 LGI1 O95972 Bone morphogeneticprotein 15 BMP15 O95994 Anterior gradient protein 2 homolog AGR2 O95998Interleukin-18-binding protein IL18BP O96009 Napsin-A NAPSA O96014Protein Wnt-11 WNT11 P00450 Ceruloplasmin CP P00451 Factor VIIIa lightchain F8 P00488 Coagulation factor XIII A chain F13A1 P00533 Epidermalgrowth factor receptor EGFR P00709 Alpha-lactalbumin LALBA P00734Prothrombin F2 P00738 Haptoglobin beta chain HP P00739Haptoglobin-related protein HPR P00740 Coagulation factor IXa heavychain F9 P00742 Factor X heavy chain F10 P00746 Complement factor D CFDP00747 Plasmin light chain B PLG P00748 Coagulation factor XIIa lightchain F12 P00749 Urokinase-type plasminogen activator long PLAU chain AP00750 Tissue-type plasminogen activator PLAT P00751 Complement factor BBa fragment CFB P00797 Renin REN P00973 2′-5′-oligoadenylate synthase 1OAS1 P00995 Pancreatic secretory trypsin inhibitor SPINK1 P01008Antithrombin-III SERPINC1 P01009 Alpha-1-antitrypsin SERPINA1 P01011Alpha-1-antichymotrypsin His-Pro-less SERPINA3 P01019 Angiotensin-1 AGTP01023 Alpha-2-macroglobulin A2M P01024 Acylation stimulating protein C3P01031 Complement C5 beta chain C5 P01033 Metalloproteinase inhibitor 1TIMP1 P01034 Cystatin-C CST3 P01036 Cystatin-S CST4 P01037 Cystatin-SNCST1 P01042 Kininogen-1 light chain KNG1 P01127 Platelet-derived growthfactor subunit B PDGFB P01135 Transforming growth factor alpha TGFAP01137 Transforming growth factor beta-1 TGFB1 P01138 Beta-nerve growthfactor NGF P01148 Gonadoliberin-1 GNRH1 P01160 Atrial natriuretic factorNPPA P01178 Oxytocin OXT P01185 Vasopressin-neurophysin 2-copeptin AVPP01189 Corticotropin POMC P01210 PENK(237-258) PENK P01213Alpha-neoendorphin PDYN P01215 Glycoprotein hormones alpha chain CGAP01222 Thyrotropin subunit beta TSHB P01225 Follitropin subunit betaFSHB P01229 Lutropin subunit beta LHB P01233 Choriogonadotropin subunitbeta CGB8 P01236 Prolactin PRL P01241 Somatotropin GH1 P01242 Growthhormone variant GH2 P01243 Chorionic somatomammotropin hormone CSH2P01258 Katacalcin CALCA P01266 Thyroglobulin TG P01270 Parathyroidhormone PTH P01275 Glucagon GCG P01282 Intestinal peptide PHM-27 VIPP01286 Somatoliberin GHRH P01298 Pancreatic prohormone PPY P01303C-flanking peptide of NPY NPY P01308 Insulin INS P01344 Insulin-likegrowth factor II IGF2 P01350 Big gastrin GAST P01374 Lymphotoxin-alphaLTA P01375 C-domain 1 TNF P01562 Interferon alpha-1/13 IFNA1 P01563Interferon alpha-2 IFNA2 P01566 Interferon alpha-10 IFNA10 P01567Interferon alpha-7 IFNA7 P01568 Interferon alpha-21 IFNA21 P01569Interferon alpha-5 IFNA5 P01570 Interferon alpha-14 IFNA14 P01571Interferon alpha-17 IFNA17 P01574 Interferon beta IFNB1 P01579Interferon gamma IFNG P01583 Interleukin-1 alpha IL1A P01584Interleukin-1 beta IL1B P01588 Erythropoietin EPO P01591 ImmunoglobulinJ chain IGJ P01732 T-cell surface glycoprotein CD8 alpha chain CD8AP01833 Polymeric immunoglobulin receptor PIGR P01857 Ig gamma-1 chain Cregion IGHG1 P01859 Ig gamma-2 chain C region IGHG2 P01860 Ig gamma-3chain C region IGHG3 P01861 Ig gamma-4 chain C region IGHG4 P01871 Ig muchain C region IGHM P01880 Ig delta chain C region IGHD P02452 Collagenalpha-1(I) chain COL1A1 P02458 Chondrocalcin COL2A1 P02461 Collagenalpha-1(III) chain COL3A1 P02462 Collagen alpha-1(IV) chain COL4A1P02647 Apolipoprotein A-I APOA1 P02649 Apolipoprotein E APOE P02652Apolipoprotein A-II APOA2 P02654 Apolipoprotein C-I APOC1 P02655Apolipoprotein C-II APOC2 P02656 Apolipoprotein C-III APOC3 P02671Fibrinogen alpha chain FGA P02675 Fibrinopeptide B FGB P02679 Fibrinogengamma chain FGG P02741 C-reactive protein CRP P02743 Serum amyloidP-component(1-203) APCS P02745 Complement C1q subcomponent subunit AC1QA P02746 Complement C1q subcomponent subunit B C1QB P02747 ComplementC1q subcomponent subunit C C1QC P02748 Complement component C9b C9P02749 Beta-2-glycoprotein 1 APOH P02750 Leucine-richalpha-2-glycoprotein LRG1 P02751 Ugl-Y2 FN1 P02753 Retinol-bindingprotein 4 RBP4 P02760 Trypstatin AMBP P02763 Alpha-1-acid glycoprotein 1ORM1 P02765 Alpha-2-HS-glycoprotein chain A AHSG P02766 TransthyretinTTR P02768 Serum albumin ALB P02771 Alpha-fetoprotein AFP P02774 VitaminD-binding protein GC P02775 Connective tissue-activating peptide IIIPPBP P02776 Platelet factor 4 PF4 P02778 CXCL10(1-73) CXCL10 P02786Transferrin receptor protein 1 TFRC P02787 Serotransferrin TF P02788Lactoferroxin-C LTF P02790 Hemopexin HPX P02808 Statherin STATH P02810Salivary acidic proline-rich phosphoprotein 1/2 PRH2 P02812 Basicsalivary proline-rich protein 2 PRB2 P02814 Peptide D1A SMR3B P02818Osteocalcin BGLAP P03950 Angiogenin ANG P03951 Coagulation factor XIaheavy chain F11 P03952 Plasma kallikrein KLKB1 P03956 27 kDainterstitial collagenase MMP1 P03971 Muellerian-inhibiting factor AMHP03973 Antileukoproteinase SLPI P04003 C4b-binding protein alpha chainC4BPA P04004 Somatomedin-B VTN P04054 Phospholipase A2 PLA2G1B P04085Platelet-derived growth factor subunit A PDGFA P04090 Relaxin A chainRLN2 P04114 Apolipoprotein B-100 APOB P04118 Colipase CLPS P04141Granulocyte-macrophage colony-stimulating CSF2 factor P04155 Trefoilfactor 1 TFF1 P04180 Phosphatidylcholine-sterol acyltransferase LCATP04196 Histidine-rich glycoprotein HRG P04217 Alpha-1B-glycoprotein A1BGP04275 von Willebrand antigen 2 VWF P04278 Sex hormone-binding globulinSHBG P04279 Alpha-inhibin-31 SEMG1 P04280 Basic salivary proline-richprotein 1 PRB1 P04628 Proto-oncogene Wnt-1 WNT1 P04745 Alpha-amylase 1AMY1A P04746 Pancreatic alpha-amylase AMY2A P04808 Prorelaxin H1 RLN1P05000 Interferon omega-1 IFNW1 P05013 Interferon alpha-6 IFNA6 P05014Interferon alpha-4 IFNA4 P05015 Interferon alpha-16 IFNA16 P05019Insulin-like growth factor I IGF1 P05060 GAWK peptide CHGB P05090Apolipoprotein D APOD P05109 Protein S100-A8 S100A8 P05111 Inhibin alphachain INHA P05112 Interleukin-4 IL4 P05113 Interleukin-5 IL5 P05120Plasminogen activator inhibitor 2 SERPINB2 P05121 Plasminogen activatorinhibitor 1 SERPINE1 P05154 Plasma serine protease inhibitor SERPINA5P05155 Plasma protease C1 inhibitor SERPING1 P05156 Complement factor Iheavy chain CFI P05160 Coagulation factor XIII B chain F13B P05161Ubiquitin-like protein ISG15 ISG15 P05230 Fibroblast growth factor 1FGF1 P05231 Interleukin-6 IL6 P05305 Big endothelin-1 EDN1 P05408C-terminal peptide SCG5 P05451 Lithostathine-1-alpha REG1A P05452Tetranectin CLEC3B P05543 Thyroxine-binding globulin SERPINA7 P05814Beta-casein CSN2 P05997 Collagen alpha-2(V) chain COL5A2 P06276Cholinesterase BCHE P06307 Cholecystokinin-12 CCK P06396 Gelsolin GSNP06681 Complement C2 C2 P06702 Protein S100-A9 S100A9 P06727Apolipoprotein A-IV APOA4 P06734 Low affinity immunoglobulin epsilon Fcreceptor FCER2 soluble form P06744 Glucose-6-phosphate isomerase GPIP06850 Corticoliberin CRH P06858 Lipoprotein lipase LPL P06881Calcitonin gene-related peptide 1 CALCA P07093 Glia-derived nexinSERPINE2 P07098 Gastric triacylglycerol lipase LIPF P07225 VitaminK-dependent protein S PROS1 P07237 Protein disulfide-isomerase P4HBP07288 Prostate-specific antigen KLK3 P07306 Asialoglycoprotein receptor1 ASGR1 P07355 Annexin A2 ANXA2 P07357 Complement component C8 alphachain C8A P07358 Complement component C8 beta chain C8B P07360Complement component C8 gamma chain C8G P07477 Alpha-trypsin chain 2PRSS1 P07478 Trypsin-2 PRSS2 P07492 Neuromedin-C GRP P07498 Kappa-caseinCSN3 P07585 Decorin DCN P07911 Uromodulin UMOD P07942 Laminin subunitbeta-1 LAMB1 P07988 Pulmonary surfactant-associated protein B SFTPBP07998 Ribonuclease pancreatic RNASE1 P08118 Beta-microseminoproteinMSMB P08123 Collagen alpha-2(I) chain COL1A2 P08185Corticosteroid-binding globulin SERPINA6 P08217 Chymotrypsin-likeelastase family member 2A CELA2A P08218 Chymotrypsin-like elastasefamily member 2B CELA2B P08253 72 kDa type IV collagenase MMP2 P08254Stromelysin-1 MMP3 P08294 Extracellular superoxide dismutase [Cu—Zn]SOD3 P08476 Inhibin beta A chain INHBA P08493 Matrix Gla protein MGPP08572 Collagen alpha-2(IV) chain COL4A2 P08581 Hepatocyte growth factorreceptor MET P08603 Complement factor H CFH P08620 Fibroblast growthfactor 4 FGF4 P08637 Low affinity immunoglobulin gamma Fc region FCGR3Areceptor III-A P08697 Alpha-2-antiplasmin SERPINF2 P08700 Interleukin-3IL3 P08709 Coagulation factor VII F7 P08833 Insulin-like growthfactor-binding protein 1 IGFBP1 P08887 Interleukin-6 receptor subunitalpha IL6R P08949 Neuromedin-B-32 NMB P08F94 Fibrocystin PKHD1 P09038Fibroblast growth factor 2 FGF2 P09228 Cystatin-SA CST2 P09237Matrilysin MMP7 P09238 Stromelysin-2 MMP10 P09341 Growth-regulated alphaprotein CXCL1 P09382 Galectin-1 LGALS1 P09466 Glycodelin PAEP P09486SPARC SPARC P09529 Inhibin beta B chain INHBB P09544 Protein Wnt-2 WNT2P09603 Processed macrophage colony-stimulating factor 1 CSF1 P09681Gastric inhibitory polypeptide GIP P09683 Secretin SCT P09919Granulocyte colony-stimulating factor CSF3 P0C091 FRAS1-relatedextracellular matrix protein 3 FREM3 P0C0L4 C4d-A C4A P0C0L5 ComplementC4-B alpha chain C4B P0C0P6 Neuropeptide S NPS P0C7L1 Serine proteaseinhibitor Kazal-type 8 SPINK8 P0C862 Complement C1q and tumor necrosisfactor- C1QTNF9 related protein 9A P0C8F1 Prostate and testis expressedprotein 4 PATE4 P0CG01 Gastrokine-3 GKN3P P0CG36 Cryptic family protein1B CFC1B P0CG37 Cryptic protein CFC1 P0CJ68 Humanin-like protein 1MTRNR2L1 P0CJ69 Humanin-like protein 2 MTRNR2L2 P0CJ70 Humanin-likeprotein 3 MTRNR2L3 P0CJ71 Humanin-like protein 4 MTRNR2L4 P0CJ72Humanin-like protein 5 MTRNR2L5 P0CJ73 Humanin-like protein 6 MTRNR2L6P0CJ74 Humanin-like protein 7 MTRNR2L7 P0CJ75 Humanin-like protein 8MTRNR2L8 P0CJ76 Humanin-like protein 9 MTRNR2L9 P0CJ77 Humanin-likeprotein 10 MTRNR2L10 P0DJD7 Pepsin A-4 PGA4 P0DJD8 Pepsin A-3 PGA3P0DJD9 Pepsin A-5 PGA5 P0DJI8 Amyloid protein A SAA1 P0DJI9 Serumamyloid A-2 protein SAA2 P10082 Peptide YY(3-36) PYY P10092 Calcitoningene-related peptide 2 CALCB P10124 Serglycin SRGN P10145 MDNCF-a IL8P10147 MIP-1-alpha(4-69) CCL3 P10163 Peptide P-D PRB4 P10451 OsteopontinSPP1 P10599 Thioredoxin TXN P10600 Transforming growth factor beta-3TGFB3 P10643 Complement component C7 C7 P10645 Vasostatin-2 CHGA P10646Tissue factor pathway inhibitor TFPI P10720 Platelet factor 4variant(4-74) PF4V1 P10745 Retinol-binding protein 3 RBP3 P10767Fibroblast growth factor 6 FGF6 P10909 Clusterin alpha chain CLU P10912Growth hormone receptor GHR P10915 Hyaluronan and proteoglycan linkprotein 1 HAPLN1 P10966 T-cell surface glycoprotein CD8 beta chain CD8BP10997 Islet amyloid polypeptide IAPP P11047 Laminin subunit gamma-1LAMC1 P11150 Hepatic triacylglycerol lipase LIPC P11226 Mannose-bindingprotein C MBL2 P11464 Pregnancy-specific beta-1-glycoprotein 1 PSG1P11465 Pregnancy-specific beta-1-glycoprotein 2 PSG2 P11487 Fibroblastgrowth factor 3 FGF3 P11597 Cholesteryl ester transfer protein CETPP11684 Uteroglobin SCGB1A1 P11686 Pulmonary surfactant-associatedprotein C SFTPC P12034 Fibroblast growth factor 5 FGF5 P12107 Collagenalpha-1(XI) chain COL11A1 P12109 Collagen alpha-1(VI) chain COL6A1P12110 Collagen alpha-2(VI) chain COL6A2 P12111 Collagen alpha-3(VI)chain COL6A3 P12259 Coagulation factor V F5 P12272 PTHrP[1-36] PTHLHP12273 Prolactin-inducible protein PIP P12544 Granzyme A GZMA P12643Bone morphogenetic protein 2 BMP2 P12644 Bone morphogenetic protein 4BMP4 P12645 Bone morphogenetic protein 3 BMP3 P12724 Eosinophil cationicprotein RNASE3 P12821 Angiotensin-converting enzyme, soluble form ACEP12838 Neutrophil defensin 4 DEFA4 P12872 Motilin MLN P13232Interleukin-7 IL7 P13236 C-C motif chemokine 4 CCL4 P13284Gamma-interferon-inducible lysosomal thiol IFI30 reductase P13500 C-Cmotif chemokine 2 CCL2 P13501 C-C motif chemokine 5 CCL5 P13521Secretogranin-2 SCG2 P13591 Neural cell adhesion molecule 1 NCAM1 P13611Versican core protein VCAN P13671 Complement component C6 C6 P13688Carcinoembryonic antigen-related cell adhesion CEACAM1 molecule 1 P13725Oncostatin-M OSM P13726 Tissue factor F3 P13727 Eosinophil granule majorbasic protein PRG2 P13942 Collagen alpha-2(XI) chain COL11A2 P13987 CD59glycoprotein CD59 P14138 Endothelin-3 EDN3 P14174 Macrophage migrationinhibitory factor MIF P14207 Folate receptor beta FOLR2 P14222Perforin-1 PRF1 P14543 Nidogen-1 NID1 P14555 Phospholipase A2, membraneassociated PLA2G2A P14625 Endoplasmin HSP90B1 P14735 Insulin-degradingenzyme IDE P14778 Interleukin-1 receptor type 1, soluble form IL1R1P14780 82 kDa matrix metalloproteinase-9 MMP9 P15018 Leukemia inhibitoryfactor LIF P15085 Carboxypeptidase A1 CPA1 P15086 Carboxypeptidase BCPB1 P15151 Poliovirus receptor PVR P15169 Carboxypeptidase N catalyticchain CPN1 P15248 Interleukin-9 IL9 P15291 N-acetyllactosamine synthaseB4GALT1 P15309 PAPf39 ACPP P15328 Folate receptor alpha FOLR1 P15374Ubiquitin carboxyl-terminal hydrolase isozyme L3 UCHL3 P15502 ElastinELN P15509 Granulocyte-macrophage colony-stimulating CSF2RA factorreceptor subunit alpha P15515 Histatin-1 HTN1 P15516His3-(31-51)-peptide HTN3 P15692 Vascular endothelial growth factor AVEGFA P15814 Immunoglobulin lambda-like polypeptide 1 IGLL1 P15907Beta-galactoside alpha-2,6-sialyltransferase 1 ST6GAL1 P15941 Mucin-1subunit beta MUC1 P16035 Metalloproteinase inhibitor 2 TIMP2 P16112Aggrecan core protein 2 ACAN P16233 Pancreatic triacylglycerol lipasePNLIP P16442 Histo-blood group ABO system transferase ABO P16471Prolactin receptor PRLR P16562 Cysteine-rich secretory protein 2 CRISP2P16619 C-C motif chemokine 3-like 1 CCL3L1 P16860 BNP(3-29) NPPB P16870Carboxypeptidase E CPE P16871 Interleukin-7 receptor subunit alpha IL7RP17213 Bactericidal permeability-increasing protein BPI P17538Chymotrypsinogen B CTRB1 P17931 Galectin-3 LGALS3 P17936 Insulin-likegrowth factor-binding protein 3 IGFBP3 P17948 Vascular endothelialgrowth factor receptor 1 FLT1 P18065 Insulin-like growth factor-bindingprotein 2 IGFBP2 P18075 Bone morphogenetic protein 7 BMP7 P18428Lipopolysaccharide-binding protein LBP P18509 PACAP-related peptideADCYAP1 P18510 Interleukin-1 receptor antagonist protein IL1RN P18827Syndecan-1 SDC1 P19021 Peptidylglycine alpha-hydroxylating PAMmonooxygenase P19235 Erythropoietin receptor EPOR P19438 Tumor necrosisfactor-binding protein 1 TNFRSF1A P19652 Alpha-1-acid glycoprotein 2ORM2 P19801 Amiloride-sensitive amine oxidase [copper- ABP1 containing]P19823 Inter-alpha-trypsin inhibitor heavy chain H2 ITIH2 P19827Inter-alpha-trypsin inhibitor heavy chain H1 ITIH1 P19835 Bilesalt-activated lipase CEL P19875 C-X-C motif chemokine 2 CXCL2 P19876C-X-C motif chemokine 3 CXCL3 P19883 Follistatin FST P19957 Elafin PI3P19961 Alpha-amylase 2B AMY2B P20061 Transcobalamin-1 TCN1 P20062Transcobalamin-2 TCN2 P20142 Gastricsin PGC P20155 Serine proteaseinhibitor Kazal-type 2 SPINK2 P20231 Tryptase beta-2 TPSB2 P20333 Tumornecrosis factor receptor superfamily TNFRSF1B member 1B P20366 SubstanceP TAC1 P20382 Melanin-concentrating hormone PMCH P20396 Thyroliberin TRHP20742 Pregnancy zone protein PZP P20774 Mimecan OGN P20783Neurotrophin-3 NTF3 P20800 Endothelin-2 EDN2 P20809 Interleukin-11 IL11P20827 Ephrin-A1 EFNA1 P20849 Collagen alpha-1(IX) chain COL9A1 P20851C4b-binding protein beta chain C4BPB P20908 Collagen alpha-1(V) chainCOL5A1 P21128 Poly(U)-specific endoribonuclease ENDOU P21246Pleiotrophin PTN P21583 Kit ligand KITLG P21741 Midkine MDK P21754 Zonapellucida sperm-binding protein 3 ZP3 P21781 Fibroblast growth factor 7FGF7 P21802 Fibroblast growth factor receptor 2 FGFR2 P21810 BiglycanBGN P21815 Bone sialoprotein 2 IBSP P21860 Receptor tyrosine-proteinkinase erbB-3 ERBB3 P21941 Cartilage matrix protein MATN1 P22003 Bonemorphogenetic protein 5 BMP5 P22004 Bone morphogenetic protein 6 BMP6P22079 Lactoperoxidase LPO P22105 Tenascin-X TNXB P22301 Interleukin-10IL10 P22303 Acetylcholinesterase ACHE P22352 Glutathione peroxidase 3GPX3 P22362 C-C motif chemokine 1 CCL1 P22455 Fibroblast growth factorreceptor 4 FGFR4 P22466 Galanin message-associated peptide GAL P22692Insulin-like growth factor-binding protein 4 IGFBP4 P22749 GranulysinGNLY P22792 Carboxypeptidase N subunit 2 CPN2 P22891 Vitamin K-dependentprotein Z PROZ P22894 Neutrophil collagenase MMP8 P23142 Fibulin-1 FBLN1P23280 Carbonic anhydrase 6 CA6 P23352 Anosmin-1 KAL1 P23435Cerebellin-1 CBLN1 P23560 Brain-derived neurotrophic factor BDNF P23582C-type natriuretic peptide NPPC P23946 Chymase CMA1 P24043 Lamininsubunit alpha-2 LAMA2 P24071 Immunoglobulin alpha Fc receptor FCARP24347 Stromelysin-3 MMP11 P24387 Corticotropin-releasing factor-bindingprotein CRHBP P24592 Insulin-like growth factor-binding protein 6 IGFBP6P24593 Insulin-like growth factor-binding protein 5 IGFBP5 P24821Tenascin TNC P24855 Deoxyribonuclease-1 DNASE1 P25067 Collagenalpha-2(VIII) chain COL8A2 P25311 Zinc-alpha-2-glycoprotein AZGP1 P25391Laminin subunit alpha-1 LAMA1 P25445 Tumor necrosis factor receptorsuperfamily FAS member 6 P25940 Collagen alpha-3(V) chain COL5A3 P25942Tumor necrosis factor receptor superfamily CD40 member 5 P26022Pentraxin-related protein PTX3 PTX3 P26927 Hepatocyte growth factor-likeprotein beta chain MST1 P27169 Serum paraoxonase/arylesterase 1 PON1P27352 Gastric intrinsic factor GIF P27487 Dipeptidyl peptidase 4membrane form DPP4 P27539 Embryonic growth/differentiation factor 1 GDF1P27658 Vastatin COL8A1 P27797 Calreticulin CALR P27918 Properdin CFPP28039 Acyloxyacyl hydrolase AOAH P28300 Protein-lysine 6-oxidase LOXP28325 Cystatin-D CST5 P28799 Granulin-1 GRN P29122 Proproteinconvertase subtilisin/kexin type 6 PCSK6 P29279 Connective tissue growthfactor CTGF P29320 Ephrin type-A receptor 3 EPHA3 P29400 Collagenalpha-5(IV) chain COL4A5 P29459 Interleukin-12 subunit alpha IL12AP29460 Interleukin-12 subunit beta IL12B P29508 Serpin B3 SERPINB3P29622 Kallistatin SERPINA4 P29965 CD40 ligand, soluble form CD40LGP30990 Neurotensin/neuromedin N NTS P31025 Lipocalin-1 LCN1 P31151Protein S100-A7 S100A7 P31371 Fibroblast growth factor 9 FGF9 P31431Syndecan-4 SDC4 P31947 14-3-3 protein sigma SFN P32455Interferon-induced guanylate-binding protein 1 GBP1 P32881 Interferonalpha-8 IFNA8 P34096 Ribonuclease 4 RNASE4 P34130 Neurotrophin-4 NTF4P34820 Bone morphogenetic protein 8B BMP8B P35030 Trypsin-3 PRSS3 P35052Secreted glypican-1 GPC1 P35070 Betacellulin BTC P35225 Interleukin-13IL13 P35247 Pulmonary surfactant-associated protein D SFTPD P35318 ADMADM P35542 Serum amyloid A-4 protein SAA4 P35555 Fibrillin-1 FBN1 P35556Fibrillin-2 FBN2 P35625 Metalloproteinase inhibitor 3 TIMP3 P35858Insulin-like growth factor-binding protein complex IGFALS acid labilesubunit P35916 Vascular endothelial growth factor receptor 3 FLT4 P35968Vascular endothelial growth factor receptor 2 KDR P36222Chitinase-3-like protein 1 CHI3L1 P36952 Serpin B5 SERPINB5 P36955Pigment epithelium-derived factor SERPINF1 P36980 Complement factorH-related protein 2 CFHR2 P39059 Collagen alpha-1(XV) chain COL15A1P39060 Collagen alpha-1(XVIII) chain COL18A1 P39877 Calcium-dependentphospholipase A2 PLA2G5 P39900 Macrophage metalloelastase MMP12 P39905Glial cell line-derived neurotrophic factor GDNF P40225 ThrombopoietinTHPO P40967 M-alpha PMEL P41159 Leptin LEP P41221 Protein Wnt-5a WNT5AP41222 Prostaglandin-H2 D-isomerase PTGDS P41271 Neuroblastomasuppressor of tumorigenicity 1 NBL1 P41439 Folate receptor gamma FOLR3P42127 Agouti-signaling protein ASIP P42702 Leukemia inhibitory factorreceptor LIFR P42830 ENA-78(9-78) CXCL5 P43026 Growth/differentiationfactor 5 GDF5 P43251 Biotinidase BTD P43652 Afamin AFM P45452Collagenase 3 MMP13 P47710 Casoxin-D CSN1S1 P47929 Galectin-7 LGALS7BP47972 Neuronal pentraxin-2 NPTX2 P47989 Xanthine oxidase XDH P47992Lymphotactin XCL1 P48023 Tumor necrosis factor ligand superfamily memberFASLG 6, membrane form P48052 Carboxypeptidase A2 CPA2 P48061 Stromalcell-derived factor 1 CXCL12 P48304 Lithostathine-1-beta REG1B P48307Tissue factor pathway inhibitor 2 TFPI2 P48357 Leptin receptor LEPRP48594 Serpin B4 SERPINB4 P48645 Neuromedin-U-25 NMU P48740Mannan-binding lectin serine protease 1 MASP1 P48745 Protein NOV homologNOV P48960 CD97 antigen subunit beta CD97 P49223 Kunitz-type proteaseinhibitor 3 SPINT3 P49747 Cartilage oligomeric matrix protein COMPP49763 Placenta growth factor PGF P49765 Vascular endothelial growthfactor B VEGFB P49767 Vascular endothelial growth factor C VEGFC P49771Fms-related tyrosine kinase 3 ligand FLT3LG P49862 Kallikrein-7 KLK7P49863 Granzyme K GZMK P49908 Selenoprotein P SEPP1 P49913 Antibacterialprotein FALL-39 CAMP P50607 Tubby protein homolog TUB P51124 Granzyme MGZMM P51512 Matrix metalloproteinase-16 MMP16 P51654 Glypican-3 GPC3P51671 Eotaxin CCL11 P51884 Lumican LUM P51888 Prolargin PRELP P52798Ephrin-A4 EFNA4 P52823 Stanniocalcin-1 STC1 P53420 Collagen alpha-4(IV)chain COL4A4 P53621 Coatomer subunit alpha COPA P54108 Cysteine-richsecretory protein 3 CRISP3 P54315 Pancreatic lipase-related protein 1PNLIPRP1 P54317 Pancreatic lipase-related protein 2 PNLIPRP2 P54793Arylsulfatase F ARSF P55000 Secreted Ly-6/uPAR-related protein 1 SLURP1P55001 Microfibrillar-associated protein 2 MFAP2 P55056 ApolipoproteinC-IV APOC4 P55058 Phospholipid transfer protein PLTP P55075 Fibroblastgrowth factor 8 FGF8 P55081 Microfibrillar-associated protein 1 MFAP1P55083 Microfibril-associated glycoprotein 4 MFAP4 P55107 Bonemorphogenetic protein 3B GDF10 P55145 Mesencephalic astrocyte-derivedneurotrophic MANF factor P55259 Pancreatic secretory granule membranemajor GP2 glycoprotein GP2 P55268 Laminin subunit beta-2 LAMB2 P55773CCL23(30-99) CCL23 P55774 C-C motif chemokine 18 CCL18 P55789 FAD-linkedsulfhydryl oxidase ALR GFER P56703 Proto-oncogene Wnt-3 WNT3 P56704Protein Wnt-3a WNT3A P56705 Protein Wnt-4 WNT4 P56706 Protein Wnt-7bWNT7B P56730 Neurotrypsin PRSS12 P56851 Epididymal secretory proteinE3-beta EDDM3B P56975 Neuregulin-3 NRG3 P58062 Serine protease inhibitorKazal-type 7 SPINK7 P58215 Lysyl oxidase homolog 3 LOXL3 P58294Prokineticin-1 PROK1 P58335 Anthrax toxin receptor 2 ANTXR2 P58397 Adisintegrin and metalloproteinase with ADAMTS12 thrombospondin motifs 12P58417 Neurexophilin-1 NXPH1 P58499 Protein FAM3B FAM3B P59510 Adisintegrin and metalloproteinase with ADAMTS20 thrombospondin motifs 20P59665 Neutrophil defensin 1 DEFA1B P59666 Neutrophil defensin 3 DEFA3P59796 Glutathione peroxidase 6 GPX6 P59826 BPI fold-containing family Bmember 3 BPIFB3 P59827 BPI fold-containing family B member 4 BPIFB4P59861 Beta-defensin 131 DEFB131 P60022 Beta-defensin 1 DEFB1 P60153Inactive ribonuclease-like protein 9 RNASE9 P60827 Complement C1q tumornecrosis factor-related C1QTNF8 protein 8 P60852 Zona pellucidasperm-binding protein 1 ZP1 P60985 Keratinocytedifferentiation-associated protein KRTDAP P61109 Kidneyandrogen-regulated protein KAP P61278 Somatostatin-14 SST P61366Osteocrin OSTN P61626 Lysozyme C LYZ P61769 Beta-2-microglobulin B2MP61812 Transforming growth factor beta-2 TGFB2 P61916 Epididymalsecretory protein E1 NPC2 P62502 Epididymal-specific lipocalin-6 LCN6P62937 Peptidyl-prolyl cis-trans isomerase A PPIA P67809Nuclease-sensitive element-binding protein 1 YBX1 P67812 Signalpeptidase complex catalytic subunit SEC11A SEC11A P78310 Coxsackievirusand adenovirus receptor CXADR P78333 Secreted glypican-5 GPC5 P78380Oxidized low-density lipoprotein receptor 1 OLR1 P78423 Processedfractalkine CX3CL1 P78509 Reelin RELN P78556 CCL20(2-70) CCL20 P80075MCP-2(6-76) CCL8 P80098 C-C motif chemokine 7 CCL7 P80108Phosphatidylinositol-glycan-specific GPLD1 phospholipase D P80162 C-X-Cmotif chemokine 6 CXCL6 P80188 Neutrophil gelatinase-associatedlipocalin LCN2 P80303 Nucleobindin-2 NUCB2 P80511 Calcitermin S100A12P81172 Hepcidin-25 HAMP P81277 Prolactin-releasing peptide PRLH P81534Beta-defensin 103 DEFB103A P81605 Dermcidin DCD P82279 Protein crumbshomolog 1 CRB1 P82987 ADAMTS-like protein 3 ADAMTSL3 P83105 Serineprotease HTRA4 HTRA4 P83110 Serine protease HTRA3 HTRA3 P83859Orexigenic neuropeptide QRFP QRFP P98088 Mucin-5AC MUC5AC P98095Fibulin-2 FBLN2 P98160 Basement membrane-specific heparan sulfate HSPG2proteoglycan core protein P98173 Protein FAM3A FAM3A Q00604 Norrin NDPQ00796 Sorbitol dehydrogenase SORD Q00887 Pregnancy-specificbeta-1-glycoprotein 9 PSG9 Q00888 Pregnancy-specific beta-1-glycoprotein4 PSG4 Q00889 Pregnancy-specific beta-1-glycoprotein 6 PSG6 Q01523HD5(56-94) DEFA5 Q01524 Defensin-6 DEFA6 Q01955 Collagen alpha-3(IV)chain COL4A3 Q02297 Pro-neuregulin-1, membrane-bound isoform NRG1 Q02325Plasminogen-like protein B PLGLB1 Q02383 Semenogelin-2 SEMG2 Q02388Collagen alpha-1(VII) chain COL7A1 Q02505 Mucin-3A MUC3A Q02509Otoconin-90 OC90 Q02747 Guanylin GUCA2A Q02763 Angiopoietin-1 receptorTEK Q02817 Mucin-2 MUC2 Q02985 Complement factor H-related protein 3CFHR3 Q03167 Transforming growth factor beta receptor type 3 TGFBR3Q03403 Trefoil factor 2 TFF2 Q03405 Urokinase plasminogen activatorsurface receptor PLAUR Q03591 Complement factor H-related protein 1CFHR1 Q03692 Collagen alpha-1(X) chain COL10A1 Q04118 Basic salivaryproline-rich protein 3 PRB3 Q04756 Hepatocyte growth factor activatorshort chain HGFAC Q04900 Sialomucin core protein 24 CD164 Q05315Eosinophil lysophospholipase CLC Q05707 Collagen alpha-1 (XIV) chainCOL14A1 Q05996 Processed zona pellucida sperm-binding protein 2 ZP2Q06033 Inter-alpha-trypsin inhibitor heavy chain H3 ITIH3 Q06141Regenerating islet-derived protein 3-alpha REG3A Q06828 FibromodulinFMOD Q07092 Collagen alpha-1(XVI) chain COL16A1 Q07325 C-X-C motifchemokine 9 CXCL9 Q07507 Dermatopontin DPT Q075Z2 Binder of spermprotein homolog 1 BSPH1 Q07654 Trefoil factor 3 TFF3 Q07699 Sodiumchannel subunit beta-1 SCN1B Q08345 Epithelial discoidindomain-containing receptor 1 DDR1 Q08380 Galectin-3-binding proteinLGALS3BP Q08397 Lysyl oxidase homolog 1 LOXL1 Q08431 Lactadherin MFGE8Q08629 Testican-1 SPOCK1 Q08648 Sperm-associated antigen 11B SPAG11BQ08830 Fibrinogen-like protein 1 FGL1 Q10471 PolypeptideN-acetylgalactosaminyltransferase 2 GALNT2 Q10472 PolypeptideN-acetylgalactosaminyltransferase 1 GALNT1 Q11201CMP-N-acetylneuraminate-beta-galactosamide- ST3GAL1alpha-2,3-sialyltransferase 1 Q11203CMP-N-acetylneuraminate-beta-1,4-galactoside ST3GAL3alpha-2,3-sialyltransferase Q11206CMP-N-acetylneuraminate-beta-galactosamide- ST3GAL4alpha-2,3-sialyltransferase 4 Q12794 Hyaluronidase-1 HYAL1 Q12805EGF-containing fibulin-like extracellular matrix EFEMP1 protein 1 Q12836Zona pellucida sperm-binding protein 4 ZP4 Q12841 Follistatin-relatedprotein 1 FSTL1 Q12904 Aminoacyl tRNA synthase complex-interacting AIMP1multifunctional protein 1 Q13018 Soluble secretory phospholipase A2receptor PLA2R1 Q13072 B melanoma antigen 1 BAGE Q13093Platelet-activating factor acetylhydrolase PLA2G7 Q13103 Secretedphosphoprotein 24 SPP2 Q13162 Peroxiredoxin-4 PRDX4 Q13201 Plateletglycoprotein Ia* MMRN1 Q13214 Semaphorin-3B SEMA3B Q13219 Pappalysin-1PAPPA Q13231 Chitotriosidase-1 CHIT1 Q13253 Noggin NOG Q13261Interleukin-15 receptor subunit alpha IL15RA Q13275 Semaphorin-3F SEMA3FQ13291 Signaling lymphocytic activation molecule SLAMF1 Q13316 Dentinmatrix acidic phosphoprotein 1 DMP1 Q13361 Microfibrillar-associatedprotein 5 MFAP5 Q13410 Butyrophilin subfamily 1 member A1 BTN1A1 Q13421Mesothelin, cleaved form MSLN Q13429 Insulin-like growth factor I IGF-IQ13443 Disintegrin and metalloproteinase domain- ADAM9 containingprotein 9 Q13519 Neuropeptide 1 PNOC Q13751 Laminin subunit beta-3 LAMB3Q13753 Laminin subunit gamma-2 LAMC2 Q13790 Apolipoprotein F APOF Q13822Ectonucleotide ENPP2 pyrophosphatase/phosphodiesterase family member 2Q14031 Collagen alpha-6(IV) chain COL4A6 Q14050 Collagen alpha-3(IX)chain COL9A3 Q14055 Collagen alpha-2(IX) chain COL9A2 Q14112 Nidogen-2NID2 Q14114 Low-density lipoprotein receptor-related protein 8 LRP8Q14118 Dystroglycan DAG1 Q14314 Fibroleukin FGL2 Q14393 Growtharrest-specific protein 6 GAS6 Q14406 Chorionic somatomammotropinhormone-like 1 CSHL1 Q14507 Epididymal secretory protein E3-alpha EDDM3AQ14508 WAP four-disulfide core domain protein 2 WFDC2 Q14512 Fibroblastgrowth factor-binding protein 1 FGFBP1 Q14515 SPARC-like protein 1SPARCL1 Q14520 Hyaluronan-binding protein 2 27 kDa light chain HABP2Q14563 Semaphorin-3A SEMA3A Q14623 Indian hedgehog protein IHH Q14624Inter-alpha-trypsin inhibitor heavy chain H4 ITIH4 Q14667 UPF0378protein KIAA0100 KIAA0100 Q14703 Membrane-bound transcription factorsite-1 MBTPS1 protease Q14766 Latent-transforming growth factorbeta-binding LTBP1 protein 1 Q14767 Latent-transforming growth factorbeta-binding LTBP2 protein 2 Q14773 Intercellular adhesion molecule 4ICAM4 Q14993 Collagen alpha-1(XIX) chain COL19A1 Q14CN2Calcium-activated chloride channel regulator 4, CLCA4 110 kDa formQ15046 Lysine--tRNA ligase KARS Q15063 Periostin POSTN Q15109 Advancedglycosylation end product-specific AGER receptor Q15113 ProcollagenC-endopeptidase enhancer 1 PCOLCE Q15166 Serum paraoxonase/lactonase 3PON3 Q15195 Plasminogen-like protein A PLGLA Q15198 Platelet-derivedgrowth factor receptor-like protein PDGFRL Q15223 Poliovirusreceptor-related protein 1 PVRL1 Q15238 Pregnancy-specificbeta-1-glycoprotein 5 PSG5 Q15363 Transmembrane emp24 domain-containingprotein 2 TMED2 Q15375 Ephrin type-A receptor 7 EPHA7 Q15389Angiopoietin-1 ANGPT1 Q15465 Sonic hedgehog protein SHH Q15485 Ficolin-2FCN2 Q15517 Corneodesmosin CDSN Q15582 Transforming growthfactor-beta-induced protein TGFBI ig-h3 Q15661 Tryptase alpha/beta-1TPSAB1 Q15726 Metastin KISS1 Q15782 Chitinase-3-like protein 2 CHI3L2Q15828 Cystatin-M CST6 Q15846 Clusterin-like protein 1 CLUL1 Q15848Adiponectin ADIPOQ Q16206 Protein disulfide-thiol oxidoreductase ENOX2Q16270 Insulin-like growth factor-binding protein 7 IGFBP7 Q16363Laminin subunit alpha-4 LAMA4 Q16378 Proline-rich protein 4 PRR4 Q16557Pregnancy-specific beta-1-glycoprotein 3 PSG3 Q16568 CART(42-89) CARTPTQ16610 Extracellular matrix protein 1 ECM1 Q16619 Cardiotrophin-1 CTF1Q16623 Syntaxin-1A STX1A Q16627 HCC-1(9-74) CCL14 Q16651 Prostasin lightchain PRSS8 Q16661 Guanylate cyclase C-activating peptide 2 GUCA2BQ16663 CCL15(29-92) CCL15 Q16674 Melanoma-derived growth regulatoryprotein MIA Q16769 Glutaminyl-peptide cyclotransferase QPCT Q16787Laminin subunit alpha-3 LAMA3 Q16842CMP-N-acetylneuraminate-beta-galactosamide- ST3GAL2alpha-2,3-sialyltransferase 2 Q17RR3 Pancreatic lipase-related protein 3PNLIPRP3 Q17RW2 Collagen alpha-1(XXIV) chain COL24A1 Q17RY6 Lymphocyteantigen 6K LY6K Q1L6U9 Prostate-associated microseminoprotein MSMPQ1W4C9 Serine protease inhibitor Kazal-type 13 SPINK13 Q1ZYL8 Izumosperm-egg fusion protein 4 IZUMO4 Q29960 HLA class I histocompatibilityantigen, Cw-16 HLA-C alpha chain Q2I0M5 R-spondin-4 RSPO4 Q2L4Q9 Serineprotease 53 PRSS53 Q2MKA7 R-spondin-1 RSPO1 Q2MV58 Tectonic-1 TCTN1Q2TAL6 Brorin VWC2 Q2UY09 Collagen alpha-1(XXVIII) chain COL28A1 Q2VPA4Complement component receptor 1-like protein CR1L Q2WEN9Carcinoembryonic antigen-related cell adhesion CEACAM16 molecule 16Q30KP8 Beta-defensin 136 DEFB136 Q30KP9 Beta-defensin 135 DEFB135 Q30KQ1Beta-defensin 133 DEFB133 Q30KQ2 Beta-defensin 130 DEFB130 Q30KQ4Beta-defensin 116 DEFB116 Q30KQ5 Beta-defensin 115 DEFB115 Q30KQ6Beta-defensin 114 DEFB114 Q30KQ7 Beta-defensin 113 DEFB113 Q30KQ8Beta-defensin 112 DEFB112 Q30KQ9 Beta-defensin 110 DEFB110 Q30KR1Beta-defensin 109 DEFB109P1 Q32P28 Prolyl 3-hydroxylase 1 LEPRE1 Q3B7J2Glucose-fructose oxidoreductase domain- GFOD2 containing protein 2Q3SY79 Protein Wnt WNT3A Q3T906 N-acetylglucosamine-1-phosphotransferaseGNPTAB subunits alpha/beta Q495T6 Membrane metallo-endopeptidase-like 1MMEL1 Q49AH0 Cerebral dopamine neurotrophic factor CDNF Q4G0G5Secretoglobin family 2B member 2 SCGB2B2 Q4G0M1 Protein FAM132B FAM132BQ4LDE5 Sushi, von Willebrand factor type A, EGF and SVEP1 pentraxindomain-containing protein 1 Q4QY38 Beta-defensin 134 DEFB134 Q4VAJ4Protein Wnt WNT10B Q4W5P6 Protein TMEM155 TMEM155 Q4ZHG4 Fibronectintype III domain-containing protein 1 FNDC1 Q53H76 Phospholipase A1member A PLA1A Q53RD9 Fibulin-7 FBLN7 Q53S33 BolA-like protein 3 BOLA3Q5BLP8 Neuropeptide-like protein C4orf48 C4orf48 Q5DT21 Serine proteaseinhibitor Kazal-type 9 SPINK9 Q5EBL8 PDZ domain-containing protein 11PDZD11 Q5FYB0 Arylsulfatase J ARSJ Q5FYB1 Arylsulfatase I ARSI Q5GAN3Ribonuclease-like protein 13 RNASE13 Q5GAN4 Ribonuclease-like protein 12RNASE12 Q5GAN6 Ribonuclease-like protein 10 RNASE10 Q5GFL6 vonWillebrand factor A domain-containing VWA2 protein 2 Q5H8A3 Neuromedin-SNMS Q5H8C1 FRAS1-related extracellular matrix protein 1 FREM1 Q5IJ48Protein crumbs homolog 2 CRB2 Q5J5C9 Beta-defensin 121 DEFB121 Q5JS37NHL repeat-containing protein 3 NHLRC3 Q5JTB6 Placenta-specific protein9 PLAC9 Q5JU69 Torsin-2A TOR2A Q5JXM2 Methyltransferase-like protein 24METTL24 Q5JZY3 Ephrin type-A receptor 10 EPHA10 Q5K4E3 Polyserase-2PRSS36 Q5SRR4 Lymphocyte antigen 6 complex locus protein G5c LY6G5CQ5T1H1 Protein eyes shut homolog EYS Q5T4F7 Secreted frizzled-relatedprotein 5 SFRP5 Q5T4W7 Artemin ARTN Q5T7M4 Protein FAM132A FAM132AQ5TEH8 Protein Wnt WNT2B Q5TIE3 von Willebrand factor Adomain-containing VWA5B1 protein 5B1 Q5UCC4 ER membrane protein complexsubunit 10 EMC10 Q5VST6 Abhydrolase domain-containing protein FAM108B1FAM108B1 Q5VTL7 Fibronectin type III domain-containing protein 7 FNDC7Q5VUM1 UPF0369 protein C6orf57 C6orf57 Q5VV43 Dyslexia-associatedprotein KIAA0319 KIAA0319 Q5VWW1 Complement C1q-like protein 3 C1QL3Q5VXI9 Lipase member N LIPN Q5VXJ0 Lipase member K LIPK Q5VXM1 CUBdomain-containing protein 2 CDCP2 Q5VYX0 Renalase RNLS Q5VYY2 Lipasemember M LIPM Q5W186 Cystatin-9 CST9 Q5W5W9 Regulated endocrine-specificprotein 18 RESP18 Q5XG92 Carboxylesterase 4A CES4A Q63HQ2 PikachurinEGFLAM Q641Q3 Meteorin-like protein METRNL Q66K79 Carboxypeptidase Z CPZQ685J3 Mucin-17 MUC17 Q68BL7 Olfactomedin-like protein 2A OLFML2A Q68BL8Olfactomedin-like protein 2B OLFML2B Q68DV7 E3 ubiquitin-protein ligaseRNF43 RNF43 Q6B9Z1 Insulin growth factor-like family member 4 IGFL4Q6BAA4 Fc receptor-like B FCRLB Q6E0U4 Dermokine DMKN Q6EMK4 VasorinVASN Q6FHJ7 Secreted frizzled-related protein 4 SFRP4 Q6GPI1Chymotrypsin B2 chain B CTRB2 Q6GTS8 Probable carboxypeptidase PM20D1PM20D1 Q6H9L7 Isthmin-2 ISM2 Q6IE36 Ovostatin homolog 2 OVOS2 Q6IE37Ovostatin homolog 1 OVOS1 Q6IE38 Serine protease inhibitor Kazal-type 14SPINK14 Q6ISS4 Leukocyte-associated immunoglobulin-like LAIR2 receptor 2Q6JVE5 Epididymal-specific lipocalin-12 LCN12 Q6JVE6 Epididymal-specificlipocalin-10 LCN10 Q6JVE9 Epididymal-specific lipocalin-8 LCN8 Q6KF10Growth/differentiation factor 6 GDF6 Q6MZW2 Follistatin-related protein4 FSTL4 Q6NSX1 Coiled-coil domain-containing protein 70 CCDC70 Q6NT32Carboxylesterase 5A CES5A Q6NT52 Choriogonadotropin subunit beta variant2 CGB2 Q6NUI6 Chondroactherin-like protein CHADL Q6NUJ1 Saposin A-likePSAPL1 Q6P093 Arylacetamide deacetylase-like 2 AADACL2 Q6P4A8Phospholipase B-like 1 PLBD1 Q6P5S2 UPF0762 protein C6orf58 C6orf58Q6P988 Protein notum homolog NOTUM Q6PCB0 von Willebrand factor Adomain-containing VWA1 protein 1 Q6PDA7 Sperm-associated antigen 11ASPAG11A Q6PEW0 Inactive serine protease 54 PRSS54 Q6PEZ8 Podocan-likeprotein 1 PODNL1 Q6PKH6 Dehydrogenase/reductase SDR family member 4-DHRS4L2 like 2 Q6Q788 Apolipoprotein A-V APOA5 Q6SPF0 Atherin SAMD1Q6UDR6 Kunitz-type protease inhibitor 4 SPINT4 Q6URK8 Testis, prostateand placenta-expressed protein TEPP Q6UW01 Cerebellin-3 CBLN3 Q6UW10Surfactant-associated protein 2 SFTA2 Q6UW15 Regenerating islet-derivedprotein 3-gamma REG3G Q6UW32 Insulin growth factor-like family member 1IGFL1 Q6UW78 UPF0723 protein C11orf83 C11orf83 Q6UW88 Epigen EPGN Q6UWE3Colipase-like protein 2 CLPSL2 Q6UWF7 NXPE family member 4 NXPE4 Q6UWF9Protein FAM180A FAM180A Q6UWM5 GLIPR1-like protein 1 GLIPR1L1 Q6UWN8Serine protease inhibitor Kazal-type 6 SPINK6 Q6UWP2Dehydrogenase/reductase SDR family member 11 DHRS11 Q6UWP8 SuprabasinSBSN Q6UWQ5 Lysozyme-like protein 1 LYZL1 Q6UWQ7 Insulin growthfactor-like family member 2 IGFL2 Q6UWR7 Ectonucleotide ENPP6pyrophosphatase/phosphodiesterase family member 6 soluble form Q6UWT2Adropin ENHO Q6UWU2 Beta-galactosidase-1-like protein GLB1L Q6UWW0Lipocalin-15 LCN15 Q6UWX4 HHIP-like protein 2 HHIPL2 Q6UWY0Arylsulfatase K ARSK Q6UWY2 Serine protease 57 PRSS57 Q6UWY5Olfactomedin-like protein 1 OLFML1 Q6UX06 Olfactomedin-4 OLFM4 Q6UX07Dehydrogenase/reductase SDR family member 13 DHRS13 Q6UX39 Amelotin AMTNQ6UX46 Protein FAM150B FAM150B Q6UX73 UPF0764 protein C16orf89 C16orf89Q6UXB0 Protein FAM131A FAM131A Q6UXB1 Insulin growth factor-like familymember 3 IGFL3 Q6UXB2 VEGF co-regulated chemokine 1 CXCL17 Q6UXF7 C-typelectin domain family 18 member B CLEC18B Q6UXH0 Hepatocellularcarcinoma-associated protein TD26 C19orf80 Q6UXH1 Cysteine-rich withEGF-like domain protein 2 CRELD2 Q6UXH8 Collagen and calcium-binding EGFdomain- CCBE1 containing protein 1 Q6UXH9 Inactive serine protease PAMR1PAMR1 Q6UXI7 Vitrin VIT Q6UXI9 Nephronectin NPNT Q6UXN2 Trem-liketranscript 4 protein TREML4 Q6UXS0 C-type lectin domain family 19 memberA CLEC19A Q6UXT8 Protein FAM150A FAM150A Q6UXT9 Abhydrolasedomain-containing protein 15 ABHD15 Q6UXV4 Apolipoprotein O-like APOOLQ6UXX5 Inter-alpha-trypsin inhibitor heavy chain H6 ITIH6 Q6UXX9R-spondin-2 RSPO2 Q6UY14 ADAMTS-like protein 4 ADAMTSL4 Q6UY27 Prostateand testis expressed protein 2 PATE2 Q6W4X9 Mucin-6 MUC6 Q6WN34Chordin-like protein 2 CHRDL2 Q6WRI0 Immunoglobulin superfamily member10 IGSF10 Q6X4U4 Sclerostin domain-containing protein 1 SOSTDC1 Q6X784Zona pellucida-binding protein 2 ZPBP2 Q6XE38 Secretoglobin family 1Dmember 4 SCGB1D4 Q6XPR3 Repetin RPTN Q6XZB0 Lipase member I LIPI Q6ZMM2ADAMTS-like protein 5 ADAMTSL5 Q6ZMP0 Thrombospondin type-1domain-containing THSD4 protein 4 Q6ZNF0 Iron/zinc purple acidphosphatase-like protein PAPL Q6ZRI0 Otogelin OTOG Q6ZRP7 Sulfhydryloxidase 2 QSOX2 Q6ZWJ8 Kielin/chordin-like protein KCP Q75N90Fibrillin-3 FBN3 Q765I0 Urotensin-2B UTS2D Q76B58 Protein FAM5C FAM5CQ76LX8 A disintegrin and metalloproteinase with ADAMTS13 thrombospondinmotifs 13 Q76M96 Coiled-coil domain-containing protein 80 CCDC80 Q7L1S5Carbohydrate sulfotransferase 9 CHST9 Q7L513 Fc receptor-like A FCRLAQ7L8A9 Vasohibin-1 VASH1 Q7RTM1 Otopetrin-1 OTOP1 Q7RTW8 Otoancorin OTOAQ7RTY5 Serine protease 48 PRSS48 Q7RTY7 Ovochymase-1 OVCH1 Q7RTZ1Ovochymase-2 OVCH2 Q7Z304 MAM domain-containing protein 2 MAMDC2 Q7Z3S9Notch homolog 2 N-terminal-like protein NOTCH2NL Q7Z4H4 Intermedin-shortADM2 Q7Z4P5 Growth/differentiation factor 7 GDF7 Q7Z4R8 UPF0669 proteinC6orf120 C6orf120 Q7Z4W2 Lysozyme-like protein 2 LYZL2 Q7Z5A4 Serineprotease 42 PRSS42 Q7Z5A7 Protein FAM19A5 FAM19A5 Q7Z5A8 Protein FAM19A3FAM19A3 Q7Z5A9 Protein FAM19A1 FAM19A1 Q7Z5J1 Hydroxysteroid11-beta-dehydrogenase 1-like HSD11B1L protein Q7Z5L0 Vitelline membraneouter layer protein 1 homolog VMO1 Q7Z5L3 Complement C1q-like protein 2C1QL2 Q7Z5L7 Podocan PODN Q7Z5P4 17-beta-hydroxysteroid dehydrogenase 13HSD17B13 Q7Z5P9 Mucin-19 MUC19 Q7Z5Y6 Bone morphogenetic protein 8ABMP8A Q7Z7B7 Beta-defensin 132 DEFB132 Q7Z7B8 Beta-defensin 128 DEFB128Q7Z7C8 Transcription initiation factor TFIID subunit 8 TAF8 Q7Z7H5Transmembrane emp24 domain-containing protein 4 TMED4 Q86SG7 Lysozymeg-like protein 2 LYG2 Q86SI9 Protein CEI C5orf38 Q86TE4 Leucine zipperprotein 2 LUZP2 Q86TH1 ADAMTS-like protein 2 ADAMTSL2 Q86U17 Serpin A11SERPINA11 Q86UU9 Endokinin-A TAC4 Q86UW8 Hyaluronan and proteoglycanlink protein 4 HAPLN4 Q86UX2 Inter-alpha-trypsin inhibitor heavy chainH5 ITIH5 Q86V24 Adiponectin receptor protein 2 ADIPOR2 Q86VB7 SolubleCD163 CD163 Q86VR8 Four-jointed box protein 1 FJX1 Q86WD7 Serpin A9SERPINA9 Q86WN2 Interferon epsilon IFNE Q86WS3 Placenta-specific 1-likeprotein PLAC1L Q86X52 Chondroitin sulfate synthase 1 CHSY1 Q86XP6Gastrokine-2 GKN2 Q86XS5 Angiopoietin-related protein 5 ANGPTL5 Q86Y27 Bmelanoma antigen 5 BAGE5 Q86Y28 B melanoma antigen 4 BAGE4 Q86Y29 Bmelanoma antigen 3 BAGE3 Q86Y30 B melanoma antigen 2 BAGE2 Q86Y38Xylosyltransferase 1 XYLT1 Q86Y78 Ly6/PLAUR domain-containing protein 6LYPD6 Q86YD3 Transmembrane protein 25 TMEM25 Q86YJ6 Threoninesynthase-like 2 THNSL2 Q86YW7 Glycoprotein hormone beta-5 GPHB5 Q86Z23Complement C1q-like protein 4 C1QL4 Q8IU57 Interleukin-28 receptorsubunit alpha IL28RA Q8IUA0 WAP four-disulfide core domain protein 8WFDC8 Q8IUB2 WAP four-disulfide core domain protein 3 WFDC3 Q8IUB3Protein WFDC10B WFDC10B Q8IUB5 WAP four-disulfide core domain protein 13WFDC13 Q8IUH2 Protein CREG2 CREG2 Q8IUK5 Plexin domain-containingprotein 1 PLXDC1 Q8IUL8 Cartilage intermediate layer protein 2 C2 CILP2Q8IUX7 Adipocyte enhancer-binding protein 1 AEBP1 Q8IUX8 Epidermalgrowth factor-like protein 6 EGFL6 Q8IVL8 Carboxypeptidase O CPO Q8IVN8Somatomedin-B and thrombospondin type-1 SBSPON domain-containing proteinQ8IVW8 Protein spinster homolog 2 SPNS2 Q8IW75 Serpin A12 SERPINA12Q8IW92 Beta-galactosidase-1-like protein 2 GLB1L2 Q8IWL1 Pulmonarysurfactant-associated protein A2 SFTPA2 Q8IWL2 Pulmonarysurfactant-associated protein A1 SFTPA1 Q8IWV2 Contactin-4 CNTN4 Q8IWY4Signal peptide, CUB and EGF-like domain- SCUBE1 containing protein 1Q8IX30 Signal peptide, CUB and EGF-like domain- SCUBE3 containingprotein 3 Q8IXA5 Sperm acrosome membrane-associated protein 3, SPACA3membrane form Q8IXB1 DnaJ homolog subfamily C member 10 DNAJC10 Q8IXL6Extracellular serine/threonine protein kinase FAM20C Fam20C Q8IYD9 Lungadenoma susceptibility protein 2 LAS2 Q8IYP2 Serine protease 58 PRSS58Q8IYS5 Osteoclast-associated immunoglobulin-like OSCAR receptor Q8IZC6Collagen alpha-1(XXVII) chain COL27A1 Q8IZJ3 C3 and PZP-likealpha-2-macroglobulin domain- CPAMD8 containing protein 8 Q8IZN7Beta-defensin 107 DEFB107B Q8N0V4 Leucine-rich repeat LGI family member2 LGI2 Q8N104 Beta-defensin 106 DEFB106B Q8N119 Matrixmetalloproteinase-21 MMP21 Q8N129 Protein canopy homolog 4 CNPY4 Q8N135Leucine-rich repeat LGI family member 4 LGI4 Q8N145 Leucine-rich repeatLGI family member 3 LGI3 Q8N158 Glypican-2 GPC2 Q8N1E2 Lysozyme g-likeprotein 1 LYG1 Q8N2E2 von Willebrand factor D and EGF domain- VWDEcontaining protein Q8N2E6 Prosalusin TOR2A Q8N2S1 Latent-transforminggrowth factor beta-binding LTBP4 protein 4 Q8N302 Angiogenic factor withG patch and FHA domains 1 AGGF1 Q8N307 Mucin-20 MUC20 Q8N323 NXPE familymember 1 NXPE1 Q8N387 Mucin-15 MUC15 Q8N3Z0 Inactive serine protease 35PRSS35 Q8N436 Inactive carboxypeptidase-like protein X2 CPXM2 Q8N474Secreted frizzled-related protein 1 SFRP1 Q8N475 Follistatin-relatedprotein 5 FSTL5 Q8N4F0 BPI fold-containing family B member 2 BPIFB2Q8N4T0 Carboxypeptidase A6 CPA6 Q8N5W8 Protein FAM24B FAM24B Q8N687Beta-defensin 125 DEFB125 Q8N688 Beta-defensin 123 DEFB123 Q8N690Beta-defensin 119 DEFB119 Q8N6C5 Immunoglobulin superfamily member 1IGSF1 Q8N6C8 Leukocyte immunoglobulin-like receptor LILRA3 subfamily Amember 3 Q8N6G6 ADAMTS-like protein 1 ADAMTSL1 Q8N6Y2 Leucine-richrepeat-containing protein 17 LRRC17 Q8N729 Neuropeptide W-23 NPW Q8N8U9BMP-binding endothelial regulator protein BMPER Q8N907 DAN domain familymember 5 DAND5 Q8NAT1 Glycosyltransferase-like domain-containing GTDC2protein 2 Q8NAU1 Fibronectin type III domain-containing protein 5 FNDC5Q8NB37 Parkinson disease 7 domain-containing protein 1 PDDC1 Q8NBI3Draxin DRAXIN Q8NBM8 Prenylcysteine oxidase-like PCYOX1L Q8NBP7Proprotein convertase subtilisin/kexin type 9 PCSK9 Q8NBQ5 Estradiol17-beta-dehydrogenase 11 HSD17B11 Q8NBV8 Synaptotagmin-8 SYT8 Q8NCC3Group XV phospholipase A2 PLA2G15 Q8NCF0 C-type lectin domain family 18member C CLEC18C Q8NCW5 NAD(P)H-hydrate epimerase APOA1BP Q8NDA2Hemicentin-2 HMCN2 Q8NDX9 Lymphocyte antigen 6 complex locus protein G5bLY6G5B Q8NDZ4 Deleted in autism protein 1 C3orf58 Q8NEB7 Acrosin-bindingprotein ACRBP Q8NES8 Beta-defensin 124 DEFB124 Q8NET1 Beta-defensin 108BDEFB108B Q8NEX5 Protein WFDC9 WFDC9 Q8NEX6 Protein WFDC11 WFDC11 Q8NF86Serine protease 33 PRSS33 Q8NFM7 Interleukin-17 receptor D IL17RD Q8NFQ5BPI fold-containing family B member 6 BPIFB6 Q8NFQ6 BPI fold-containingfamily C protein BPIFC Q8NFU4 Follicular dendritic cell secreted peptideFDCSP Q8NFW1 Collagen alpha-1(XXII) chain COL22A1 Q8NG35 Beta-defensin105 DEFB105B Q8NG41 Neuropeptide B-23 NPB Q8NHW6 Otospiralin OTOS Q8NI99Angiopoietin-related protein 6 ANGPTL6 Q8TAA1 Probable ribonuclease 11RNASE11 Q8TAG5 V-set and transmembrane domain-containing VSTM2A protein2A Q8TAL6 Fin bud initiation factor homolog FIBIN Q8TAT2 Fibroblastgrowth factor-binding protein 3 FGFBP3 Q8TAX7 Mucin-7 MUC7 Q8TB22Spermatogenesis-associated protein 20 SPATA20 Q8TB73 Protein NDNF NDNFQ8TB96 T-cell immunomodulatory protein ITFG1 Q8TC92 Proteindisulfide-thiol oxidoreductase ENOX1 Q8TCV5 WAP four-disulfide coredomain protein 5 WFDC5 Q8TD06 Anterior gradient protein 3 homolog AGR3Q8TD33 Secretoglobin family 1C member 1 SCGB1C1 Q8TD46 Cell surfaceglycoprotein CD200 receptor 1 CD200R1 Q8TDE3 Ribonuclease 8 RNASE8Q8TDF5 Neuropilin and tolloid-like protein 1 NETO1 Q8TDL5 BPIfold-containing family B member 1 BPIFB1 Q8TE56 A disintegrin andmetalloproteinase with ADAMTS17 thrombospondin motifs 17 Q8TE57 Adisintegrin and metalloproteinase with ADAMTS16 thrombospondin motifs 16Q8TE58 A disintegrin and metalloproteinase with ADAMTS15 thrombospondinmotifs 15 Q8TE59 A disintegrin and metalloproteinase with ADAMTS19thrombospondin motifs 19 Q8TE60 A disintegrin and metalloproteinase withADAMTS18 thrombospondin motifs 18 Q8TE99 Acid phosphatase-like protein 2ACPL2 Q8TER0 Sushi, nidogen and EGF-like domain-containing SNED1 protein1 Q8TEU8 WAP, kazal, immunoglobulin, kunitz and NTR WFIKKN2domain-containing protein 2 Q8WTQ1 Beta-defensin 104 DEFB104B Q8WTR8Netrin-5 NTN5 Q8WTU2 Scavenger receptor cysteine-rich domain- SRCRB4Dcontaining group B protein Q8WU66 Protein TSPEAR TSPEAR Q8WUA8 TsukushinTSKU Q8WUF8 Protein FAM172A FAM172A Q8WUJ1 Neuferricin CYB5D2 Q8WUY1UPF0670 protein THEM6 THEM6 Q8WVN6 Secreted and transmembrane protein 1SECTM1 Q8WVQ1 Soluble calcium-activated nucleotidase 1 CANT1 Q8WWA0Intelectin-1 ITLN1 Q8WWG1 Neuregulin-4 NRG4 Q8WWQ2 Inactive heparanase-2HPSE2 Q8WWU7 Intelectin-2 ITLN2 Q8WWY7 WAP four-disulfide core domainprotein 12 WFDC12 Q8WWY8 Lipase member H LIPH Q8WWZ8 Oncoprotein-inducedtranscript 3 protein OIT3 Q8WX39 Epididymal-specific lipocalin-9 LCN9Q8WXA2 Prostate and testis expressed protein 1 PATE1 Q8WXD2Secretogranin-3 SCG3 Q8WXF3 Relaxin-3 A chain RLN3 Q8WXI7 Mucin-16 MUC16Q8WXQ8 Carboxypeptidase A5 CPA5 Q8WXS8 A disintegrin andmetalloproteinase with ADAMTS14 thrombospondin motifs 14 Q92484 Acidsphingomyelinase-like phosphodiesterase 3a SMPDL3A Q92485 Acidsphingomyelinase-like phosphodiesterase 3b SMPDL3B Q92496 Complementfactor H-related protein 4 CFHR4 Q92520 Protein FAM3C FAM3C Q92563Testican-2 SPOCK2 Q92583 C-C motif chemokine 17 CCL17 Q92626 Peroxidasinhomolog PXDN Q92743 Serine protease HTRA1 HTRA1 Q92752 Tenascin-R TNRQ92765 Secreted frizzled-related protein 3 FRZB Q92819 Hyaluronansynthase 2 HAS2 Q92820 Gamma-glutamyl hydrolase GGH Q92824 Proproteinconvertase subtilisin/kexin type 5 PCSK5 Q92832 Protein kinase C-bindingprotein NELL1 NELL1 Q92838 Ectodysplasin-A, membrane form EDA Q92874Deoxyribonuclease-1-like 2 DNASE1L2 Q92876 Kallikrein-6 KLK6 Q92913Fibroblast growth factor 13 FGF13 Q92954 Proteoglycan 4 C-terminal partPRG4 Q93038 Tumor necrosis factor receptor superfamily TNFRSF25 member25 Q93091 Ribonuclease K6 RNASE6 Q93097 Protein Wnt-2b WNT2B Q93098Protein Wnt-8b WNT8B Q95460 Major histocompatibility complex classI-related MR1 gene protein Q969D9 Thymic stromal lymphopoietin TSLPQ969E1 Liver-expressed antimicrobial peptide 2 LEAP2 Q969H8 UPF0556protein C19orf10 C19orf10 Q969Y0 NXPE family member 3 NXPE3 Q96A54Adiponectin receptor protein 1 ADIPOR1 Q96A83 Collagen alpha-1(XXVI)chain EMID2 Q96A84 EMI domain-containing protein 1 EMID1 Q96A98Tuberoinfundibular peptide of 39 residues PTH2 Q96A99 Pentraxin-4 PTX4Q96BH3 Epididymal sperm-binding protein 1 ELSPBP1 Q96BQ1 Protein FAM3DFAM3D Q96CG8 Collagen triple helix repeat-containing protein 1 CTHRC1Q96DA0 Zymogen granule protein 16 homolog B ZG16B Q96DN2 von Willebrandfactor C and EGF domain- VWCE containing protein Q96DR5 BPIfold-containing family A member 2 BPIFA2 Q96DR8 Mucin-like protein 1MUCL1 Q96DX4 RING finger and SPRY domain-containing protein 1 RSPRY1Q96EE4 Coiled-coil domain-containing protein 126 CCDC126 Q96GS6Abhydrolase domain-containing protein FAM108A1 FAM108A1 Q96GW7 Brevicancore protein BCAN Q96HF1 Secreted frizzled-related protein 2 SFRP2Q96I82 Kazal-type serine protease inhibitor domain- KAZALD1 containingprotein 1 Q96ID5 Immunoglobulin superfamily member 21 IGSF21 Q96II8Leucine-rich repeat and calponin homology LRCH3 domain-containingprotein 3 Q96IY4 Carboxypeptidase B2 CPB2 Q96JB6 Lysyl oxidase homolog 4LOXL4 Q96JK4 HHIP-like protein 1 HHIPL1 Q96KN2 Beta-Ala-His dipeptidaseCNDP1 Q96KW9 Protein SPACA7 SPACA7 Q96KX0 Lysozyme-like protein 4 LYZL4Q96L15 Ecto-ADP-ribosyltransferase 5 ART5 Q96LB8 Peptidoglycanrecognition protein 4 PGLYRP4 Q96LB9 Peptidoglycan recognition protein 3PGLYRP3 Q96LC7 Sialic acid-binding Ig-like lectin 10 SIGLEC10 Q96LR4Protein FAM19A4 FAM19A4 Q96MK3 Protein FAM20A FAM20A Q96MS3Glycosyltransferase 1 domain-containing protein 1 GLT1D1 Q96NY8Processed poliovirus receptor-related protein 4 PVRL4 Q96NZ8 WAP, kazal,immunoglobulin, kunitz and NTR WFIKKN1 domain-containing protein 1Q96NZ9 Proline-rich acidic protein 1 PRAP1 Q96P44 Collagen alpha-1(XXI)chain COL21A1 Q96PB7 Noelin-3 OLFM3 Q96PC5 Melanoma inhibitory activityprotein 2 MIA2 Q96PD5 N-acetylmuramoyl-L-alanine amidase PGLYRP2 Q96PH6Beta-defensin 118 DEFB118 Q96PL1 Secretoglobin family 3A member 2SCGB3A2 Q96PL2 Beta-tectorin TECTB Q96QH8 Sperm acrosome-associatedprotein 5 SPACA5 Q96QR1 Secretoglobin family 3A member 1 SCGB3A1 Q96QU1Protocadherin-15 PCDH15 Q96QV1 Hedgehog-interacting protein HHIP Q96RW7Hemicentin-1 HMCN1 Q96S42 Nodal homolog NODAL Q96S86 Hyaluronan andproteoglycan link protein 3 HAPLN3 Q96SL4 Glutathione peroxidase 7 GPX7Q96SM3 Probable carboxypeptidase X1 CPXM1 Q96T91 Glycoprotein hormonealpha-2 GPHA2 Q99062 Granulocyte colony-stimulating factor receptorCSF3R Q99102 Mucin-4 alpha chain MUC4 Q99217 Amelogenin, X isoform AMELXQ99218 Amelogenin, Y isoform AMELY Q99435 Protein kinase C-bindingprotein NELL2 NELL2 Q99470 Stromal cell-derived factor 2 SDF2 Q99542Matrix metalloproteinase-19 MMP19 Q99574 Neuroserpin SERPINI1 Q99584Protein S100-A13 S100A13 Q99616 C-C motif chemokine 13 CCL13 Q99645Epiphycan EPYC Q99674 Cell growth regulator with EF hand domain CGREF1protein 1 Q99715 Collagen alpha-1(XII) chain COL12A1 Q99727Metalloproteinase inhibitor 4 TIMP4 Q99731 C-C motif chemokine 19 CCL19Q99748 Neurturin NRTN Q99935 Proline-rich protein 1 PROL1 Q99942 E3ubiquitin-protein ligase RNF5 RNF5 Q99944 Epidermal growth factor-likeprotein 8 EGFL8 Q99954 Submaxillary gland androgen-regulated protein 3ASMR3A Q99969 Retinoic acid receptor responder protein 2 RARRES2 Q99972Myocilin MYOC Q99983 Osteomodulin OMD Q99985 Semaphorin-3C SEMA3C Q99988Growth/differentiation factor 15 GDF15 Q9BPW4 Apolipoprotein L4 APOL4Q9BQ08 Resistin-like beta RETNLB Q9BQ16 Testican-3 SPOCK3 Q9BQ51Programmed cell death 1 ligand 2 PDCD1LG2 Q9BQB4 Sclerostin SOST Q9BQI4Coiled-coil domain-containing protein 3 CCDC3 Q9BQP9 BPI fold-containingfamily A member 3 BPIFA3 Q9BQR3 Serine protease 27 PRSS27 Q9BQY6 WAPfour-disulfide core domain protein 6 WFDC6 Q9BRR6 ADP-dependentglucokinase ADPGK Q9BS86 Zona pellucida-binding protein 1 ZPBP Q9BSG0Protease-associated domain-containing protein 1 PRADC1 Q9BSG5 RetbindinRTBDN Q9BT30 Probable alpha-ketoglutarate-dependent ALKBH7 dioxygenaseABH7 Q9BT56 Spexin C12orf39 Q9BT67 NEDD4 family-interacting protein 1NDFIP1 Q9BTY2 Plasma alpha-L-fucosidase FUCA2 Q9BU40 Chordin-likeprotein 1 CHRDL1 Q9BUD6 Spondin-2 SPON2 Q9BUN1 Protein MENT MENT Q9BUR5Apolipoprotein O APOO Q9BV94 ER degradation-enhancingalpha-mannosidase-like 2 EDEM2 Q9BWP8 Collectin-11 COLEC11 Q9BWS9Chitinase domain-containing protein 1 CHID1 Q9BX67 Junctional adhesionmolecule C JAM3 Q9BX93 Group XIIB secretory phospholipase A2-likePLA2G12B protein Q9BXI9 Complement C1q tumor necrosis factor-relatedC1QTNF6 protein 6 Q9BXJ0 Complement C1q tumor necrosis factor-relatedC1QTNF5 protein 5 Q9BXJ1 Complement C1q tumor necrosis factor-relatedC1QTNF1 protein 1 Q9BXJ2 Complement C1q tumor necrosis factor-relatedC1QTNF7 protein 7 Q9BXJ3 Complement C1q tumor necrosis factor-relatedC1QTNF4 protein 4 Q9BXJ4 Complement C1q tumor necrosis factor-relatedC1QTNF3 protein 3 Q9BXJ5 Complement C1q tumor necrosis factor-relatedC1QTNF2 protein 2 Q9BXN1 Asporin ASPN Q9BXP8 Pappalysin-2 PAPPA2 Q9BXR6Complement factor H-related protein 5 CFHR5 Q9BXS0 Collagen alpha-1(XXV)chain COL25A1 Q9BXX0 EMILIN-2 EMILIN2 Q9BXY4 R-spondin-3 RSPO3 Q9BY15EGF-like module-containing mucin-like hormone EMR3 receptor-like 3subunit beta Q9BY50 Signal peptidase complex catalytic subunit SEC11CSEC11C Q9BY76 Angiopoietin-related protein 4 ANGPTL4 Q9BYF1 Processedangiotensin-converting enzyme 2 ACE2 Q9BYJ0 Fibroblast growthfactor-binding protein 2 FGFBP2 Q9BYW3 Beta-defensin 126 DEFB126 Q9BYX4Interferon-induced helicase C domain-containing IFIH1 protein 1 Q9BYZ8Regenerating islet-derived protein 4 REG4 Q9BZ76 Contactin-associatedprotein-like 3 CNTNAP3 Q9BZG9 Ly-6/neurotoxin-like protein 1 LYNX1Q9BZJ3 Tryptase delta TPSD1 Q9BZM1 Group XIIA secretory phospholipase A2PLA2G12A Q9BZM2 Group IIF secretory phospholipase A2 PLA2G2F Q9BZM5NKG2D ligand 2 ULBP2 Q9BZP6 Acidic mammalian chitinase CHIA Q9BZZ2Sialoadhesin SIGLEC1 Q9C0B6 Protein FAM5B FAM5B Q9GZM7Tubulointerstitial nephritis antigen-like TINAGL1 Q9GZN4 Brain-specificserine protease 4 PRSS22 Q9GZP0 Platelet-derived growth factor D,receptor-binding PDGFD form Q9GZT5 Protein Wnt-10a WNT10A Q9GZU5Nyctalopin NYX Q9GZV7 Hyaluronan and proteoglycan link protein 2 HAPLN2Q9GZV9 Fibroblast growth factor 23 FGF23 Q9GZX9 Twisted gastrulationprotein homolog 1 TWSG1 Q9GZZ7 GDNF family receptor alpha-4 GFRA4 Q9GZZ8Extracellular glycoprotein lacritin LACRT Q9H0B8 Cysteine-rich secretoryprotein LCCL domain- CRISPLD2 containing 2 Q9H106 Signal-regulatoryprotein delta SIRPD Q9H114 Cystatin-like 1 CSTL1 Q9H173 Nucleotideexchange factor SIL1 SIL1 Q9H1E1 Ribonuclease 7 RNASE7 Q9H1F0 WAPfour-disulfide core domain protein 10A WFDC10A Q9H1J5 Protein Wnt-8aWNT8A Q9H1J7 Protein Wnt-5b WNT5B Q9H1M3 Beta-defensin 129 DEFB129Q9H1M4 Beta-defensin 127 DEFB127 Q9H1Z8 Augurin C2orf40 Q9H239 Matrixmetalloproteinase-28 MMP28 Q9H2A7 C-X-C motif chemokine 16 CXCL16 Q9H2A9Carbohydrate sulfotransferase 8 CHST8 Q9H2R5 Kallikrein-15 KLK15 Q9H2X0Chordin CHRD Q9H2X3 C-type lectin domain family 4 member M CLEC4M Q9H306Matrix metalloproteinase-27 MMP27 Q9H324 A disintegrin andmetalloproteinase with ADAMTS10 thrombospondin motifs 10 Q9H336Cysteine-rich secretory protein LCCL domain- CRISPLD1 containing 1Q9H3E2 Sorting nexin-25 SNX25 Q9H3R2 Mucin-13 MUC13 Q9H3U7 SPARC-relatedmodular calcium-binding protein 2 SMOC2 Q9H3Y0 Peptidase inhibitorR3HDML R3HDML Q9H4A4 Aminopeptidase B RNPEP Q9H4F8 SPARC-related modularcalcium-binding protein 1 SMOC1 Q9H4G1 Cystatin-9-like CST9L Q9H5V8 CUBdomain-containing protein 1 CDCP1 Q9H6B9 Epoxide hydrolase 3 EPHX3Q9H6E4 Coiled-coil domain-containing protein 134 CCDC134 Q9H741 UPF0454protein C12orf49 C12orf49 Q9H772 Gremlin-2 GREM2 Q9H7Y0 Deleted inautism-related protein 1 CXorf36 Q9H8L6 Multimerin-2 MMRN2 Q9H9S5Fukutin-related protein FKRP Q9HAT2 Sialate O-acetylesterase SIAE Q9HB40Retinoid-inducible serine carboxypeptidase SCPEP1 Q9HB63 Netrin-4 NTN4Q9HBJ0 Placenta-specific protein 1 PLAC1 Q9HC23 Prokineticin-2 PROK2Q9HC57 WAP four-disulfide core domain protein 1 WFDC1 Q9HC73 Cytokinereceptor-like factor 2 CRLF2 Q9HC84 Mucin-5B MUC5B Q9HCB6 Spondin-1SPON1 Q9HCQ7 Neuropeptide NPSF NPVF Q9HCT0 Fibroblast growth factor 22FGF22 Q9HD89 Resistin RETN Q9NNX1 Tuftelin TUFT1 Q9NNX6 CD209 antigenCD209 Q9NP55 BPI fold-containing family A member 1 BPIFA1 Q9NP70Ameloblastin AMBN Q9NP95 Fibroblast growth factor 20 FGF20 Q9NP99Triggering receptor expressed on myeloid cells 1 TREM1 Q9NPA2 Matrixmetalloproteinase-25 MMP25 Q9NPE2 Neugrin NGRN Q9NPH0 Lysophosphatidicacid phosphatase type 6 ACP6 Q9NPH6 Odorant-binding protein 2b OBP2BQ9NQ30 Endothelial cell-specific molecule 1 ESM1 Q9NQ36 Signal peptide,CUB and EGF-like domain- SCUBE2 containing protein 2 Q9NQ38 Serineprotease inhibitor Kazal-type 5 SPINK5 Q9NQ76 Matrix extracellularphosphoglycoprotein MEPE Q9NQ79 Cartilage acidic protein 1 CRTAC1 Q9NR16Scavenger receptor cysteine-rich type 1 protein CD163L1 M160 Q9NR23Growth/differentiation factor 3 GDF3 Q9NR71 Neutral ceramidase ASAH2Q9NR99 Matrix-remodeling-associated protein 5 MXRA5 Q9NRA1Platelet-derived growth factor C PDGFC Q9NRC9 Otoraplin OTOR Q9NRE1Matrix metalloproteinase-26 MMP26 Q9NRJ3 C-C motif chemokine 28 CCL28Q9NRM1 Enamelin ENAM Q9NRN5 Olfactomedin-like protein 3 OLFML3 Q9NRR1Cytokine-like protein 1 CYTL1 Q9NS15 Latent-transforming growth factorbeta-binding LTBP3 protein 3 Q9NS62 Thrombospondin type-1domain-containing THSD1 protein 1 Q9NS71 Gastrokine-1 GKN1 Q9NS98Semaphorin-3G SEMA3G Q9NSA1 Fibroblast growth factor 21 FGF21 Q9NT22EMILIN-3 EMILIN3 Q9NTU7 Cerebellin-4 CBLN4 Q9NVR0 Kelch-like protein 11KLHL11 Q9NWH7 Spermatogenesis-associated protein 6 SPATA6 Q9NXC2Glucose-fructose oxidoreductase domain- GFOD1 containing protein 1Q9NY56 Odorant-binding protein 2a OBP2A Q9NY84 Vascular non-inflammatorymolecule 3 VNN3 Q9NZ20 Group 3 secretory phospholipase A2 PLA2G3 Q9NZC2Triggering receptor expressed on myeloid cells 2 TREM2 Q9NZK5 Adenosinedeaminase CECR1 CECR1 Q9NZK7 Group IIE secretory phospholipase A2PLA2G2E Q9NZP8 Complement C1r subcomponent-like protein C1RL Q9NZV1Cysteine-rich motor neuron 1 protein CRIM1 Q9NZW4 Dentin sialoproteinDSPP Q9P0G3 Kallikrein-14 KLK14 Q9P0W0 Interferon kappa IFNK Q9P218Collagen alpha-1(XX) chain COL20A1 Q9P2C4 Transmembrane protein 181TMEM181 Q9P2K2 Thioredoxin domain-containing protein 16 TXNDC16 Q9P2N4 Adisintegrin and metalloproteinase with ADAMTS9 thrombospondin motifs 9Q9UBC7 Galanin-like peptide GALP Q9UBD3 Cytokine SCM-1 beta XCL2 Q9UBD9Cardiotrophin-like cytokine factor 1 CLCF1 Q9UBM4 Opticin OPTC Q9UBP4Dickkopf-related protein 3 DKK3 Q9UBQ6 Exostosin-like 2 EXTL2 Q9UBR5Chemokine-like factor CKLF Q9UBS5 Gamma-aminobutyric acid type Breceptor subunit GABBR1 1 Q9UBT3 Dickkopf-related protein 4 short formDKK4 Q9UBU2 Dickkopf-related protein 2 DKK2 Q9UBU3 Ghrelin-28 GHRLQ9UBV4 Protein Wnt-16 WNT16 Q9UBX5 Fibulin-5 FBLN5 Q9UBX7 Kallikrein-11KLK11 Q9UEF7 Klotho KL Q9UFP1 Protein FAM198A FAM198A Q9UGM3 Deleted inmalignant brain tumors 1 protein DMBT1 Q9UGM5 Fetuin-B FETUB Q9UGP8Translocation protein SEC63 homolog SEC63 Q9UHF0 Neurokinin-B TAC3Q9UHF1 Epidermal growth factor-like protein 7 EGFL7 Q9UHG2 ProSAASPCSK1N Q9UHI8 A disintegrin and metalloproteinase with ADAMTS1thrombospondin motifs 1 Q9UHL4 Dipeptidyl peptidase 2 DPP7 Q9UI42Carboxypeptidase A4 CPA4 Q9UIG4 Psoriasis susceptibility 1 candidategene 2 protein PSORS1C2 Q9UIK5 Tomoregulin-2 TMEFF2 Q9UIQ6Leucyl-cystinyl aminopeptidase, pregnancy serum LNPEP form Q9UJA9Ectonucleotide ENPP5 pyrophosphatase/phosphodiesterase family member 5Q9UJH8 Meteorin METRN Q9UJJ9 N-acetylglucosamine-1-phosphotransferaseGNPTG subunit gamma Q9UJW2 Tubulointerstitial nephritis antigen TINAGQ9UK05 Growth/differentiation factor 2 GDF2 Q9UK55 Protein Z-dependentprotease inhibitor SERPINA10 Q9UK85 Dickkopf-like protein 1 DKKL1 Q9UKJ1Paired immunoglobulin-like type 2 receptor alpha PILRA Q9UKP4 Adisintegrin and metalloproteinase with ADAMTS7 thrombospondin motifs 7Q9UKP5 A disintegrin and metalloproteinase with ADAMTS6 thrombospondinmotifs 6 Q9UKQ2 Disintegrin and metalloproteinase domain- ADAM28containing protein 28 Q9UKQ9 Kallikrein-9 KLK9 Q9UKR0 Kallikrein-12KLK12 Q9UKR3 Kallikrein-13 KLK13 Q9UKU9 Angiopoietin-related protein 2ANGPTL2 Q9UKZ9 Procollagen C-endopeptidase enhancer 2 PCOLCE2 Q9UL52Transmembrane protease serine 11E non-catalytic TMPRSS11E chain Q9ULC0Endomucin EMCN Q9ULI3 Protein HEG homolog 1 HEG1 Q9ULZ1 Apelin-13 APLNQ9ULZ9 Matrix metalloproteinase-17 MMP17 Q9UM21Alpha-1,3-mannosyl-glycoprotein 4-beta-N- MGAT4Aacetylglucosaminyltransferase A soluble form Q9UM22 Mammalianependymin-related protein 1 EPDR1 Q9UM73 ALK tyrosine kinase receptorALK Q9UMD9 97 kDa linear IgA disease antigen COL17A1 Q9UMX5 NeudesinNENF Q9UN73 Protocadherin alpha-6 PCDHA6 Q9UNA0 A disintegrin andmetalloproteinase with ADAMTS5 thrombospondin motifs 5 Q9UNI1Chymotrypsin-like elastase family member 1 CELA1 Q9UNK4 Group IIDsecretory phospholipase A2 PLA2G2D Q9UP79 A disintegrin andmetalloproteinase with ADAMTS8 thrombospondin motifs 8 Q9UPZ6Thrombospondin type-1 domain-containing THSD7A protein 7A Q9UQ72Pregnancy-specific beta-1-glycoprotein 11 PSG11 Q9UQ74Pregnancy-specific beta-1-glycoprotein 8 PSG8 Q9UQC9 Calcium-activatedchloride channel regulator 2 CLCA2 Q9UQE7 Structural maintenance ofchromosomes protein 3 SMC3 Q9UQP3 Tenascin-N TNN Q9Y223UDP-N-acetylglucosamine 2-epimerase GNE Q9Y240 C-type lectin domainfamily 11 member A CLEC11A Q9Y251 Heparanase 8 kDa subunit HPSE Q9Y258C-C motif chemokine 26 CCL26 Q9Y264 Angiopoietin-4 ANGPT4 Q9Y275 Tumornecrosis factor ligand superfamily member TNFSF13B 13b, membrane formQ9Y287 BRI2 intracellular domain ITM2B Q9Y2E5 Epididymis-specificalpha-mannosidase MAN2B2 Q9Y334 von Willebrand factor Adomain-containing VWA7 protein 7 Q9Y337 Kallikrein-5 KLK5 Q9Y3B3Transmembrane emp24 domain-containing protein 7 TMED7 Q9Y3E2 BolA-likeprotein 1 BOLA1 Q9Y426 C2 domain-containing protein 2 C2CD2 Q9Y4K0 Lysyloxidase homolog 2 LOXL2 Q9Y4X3 C-C motif chemokine 27 CCL27 Q9Y5C1Angiopoietin-related protein 3 ANGPTL3 Q9Y5I2 Protocadherin alpha-10PCDHA10 Q9Y5I3 Protocadherin alpha-1 PCDHA1 Q9Y5K2 Kallikrein-4 KLK4Q9Y5L2 Hypoxia-inducible lipid droplet-associated protein HILPDA Q9Y5Q5Atrial natriuretic peptide-converting enzyme CORIN Q9Y5R2 Matrixmetalloproteinase-24 MMP24 Q9Y5U5 Tumor necrosis factor receptorsuperfamily TNFRSF18 member 18 Q9Y5W5 Wnt inhibitory factor 1 WIF1Q9Y5X9 Endothelial lipase LIPG Q9Y625 Secreted glypican-6 GPC6 Q9Y646Carboxypeptidase Q CPQ Q9Y6C2 EMILIN-1 EMILIN1 Q9Y6F9 Protein Wnt-6 WNT6Q9Y6I9 Testis-expressed sequence 264 protein TEX264 Q9Y6L7 Tolloid-likeprotein 2 TLL2 Q9Y6N3 Calcium-activated chloride channel regulatorCLCA3P family member 3 Q9Y6N6 Laminin subunit gamma-3 LAMC3 Q9Y6R7IgGFc-binding protein FCGBP Q9Y6Y9 Lymphocyte antigen 96 LY96 Q9Y6Z7Collectin-10 COLEC10

The Uniprot IDs set forth in Table 1 refer to the human versions thelisted proteins and the sequences of each are available from the Uniprotdatabase. Sequences of the listed proteins are also generally availablefor various animals, including various mammals and animals of veterinaryor industrial interest. Accordingly, in some embodiments, compositionsand methods of the invention provide for the delivery of one or moremRNAs encoding one or more proteins chosen from mammalian homologs orhomologs from an animal of veterinary or industrial interest of thesecreted proteins listed in Table 1; thus, compositions of the inventionmay comprise an mRNA encoding a protein chosen from mammalian homologsor homologs from an animal of veterinary or industrial interest of aprotein listed in Table 1 along with other components set out herein,and methods of the invention may comprise preparing and/or administeringa composition comprising an mRNA encoding a protein chosen frommammalian homologs or homologs from an animal of veterinary orindustrial interest of a protein listed in Table 1 along with othercomponents set out herein. In some embodiments, mammalian homologs arechosen from mouse, rat, hamster, gerbil, horse, pig, cow, llama, alpaca,mink, dog, cat, ferret, sheep, goat, or camel homologs. In someembodiments, the animal of veterinary or industrial interest is chosenfrom the mammals listed above and/or chicken, duck, turkey, salmon,catfish, or tilapia.

In some embodiments, the compositions and methods of the inventionprovide for the delivery of one or more mRNAs encoding one or moreadditional exemplary proteins listed in Table 2; thus, compositions ofthe invention may comprise an mRNA encoding a protein listed in Table 2(or a homolog thereof, as discussed below) along with other componentsset out herein, and methods of the invention may comprise preparingand/or administering a composition comprising an mRNA encoding a proteinchosen from the proteins listed in Table 2 (or a homolog thereof, asdiscussed below) along with other components set out herein.

TABLE 2 Additional Exemplary Proteins Uniprot ID Protein Name Gene NameA6NGW2 Putative stereocilin-like protein STRCP1 A6NIE9 Putative serineprotease 29 PRSS29P A6NJ16 Putative V-set and immunoglobulin IGHV4OR15-8domain-containing-like protein IGHV4OR15-8 A6NJS3 Putative V-set andimmunoglobulin IGHV1OR21-1 domain-containing-like protein IGHV1OR21-1A6NMY6 Putative annexin A2-like protein ANXA2P2 A8MT79 Putativezinc-alpha-2-glycoprotein-like 1 A8MWS1 Putative killer cellimmunoglobulin-like KIR3DP1 receptor like protein KIR3DP1 A8MXU0Putative beta-defensin 108A DEFB108P1 C9JUS6 Putativeadrenomedullin-5-like protein ADM5 P0C7V7 Putative signal peptidasecomplex SEC11B catalytic subunit SEC11B P0C854 Putative cat eye syndromecritical region CECR9 protein 9 Q13046 Putative pregnancy-specificbeta-1- PSG7 glycoprotein 7 Q16609 Putative apolipoprotein(a)-likeprotein 2 LPAL2 Q2TV78 Putative macrophage-stimulating protein MST1P9MSTP9 Q5JQD4 Putative peptide YY-3 PYY3 Q5R387 Putative inactive groupIIC secretory PLA2G2C phospholipase A2 Q5VSP4 Putative lipocalin 1-likeprotein 1 LCN1P1 Q5W188 Putative cystatin-9-like protein CST9LP1 CST9LP1Q6UXR4 Putative serpin A13 SERPINA13P Q86SH4 Putative testis-specificprion protein PRNT Q86YQ2 Putative latherin LATH Q8IVG9 Putative humaninpeptide MT-RNR2 Q8NHM4 Putative trypsin-6 TRY6 Q8NHW4 C-C motifchemokine 4-like CCL4L2 Q9H7L2 Putative killer cell immunoglobulin-likeKIR3DX1 receptor-like protein KIR3DX1 Q9NRI6 Putative peptide YY-2 PYY2Q9UF72 Putative TP73 antisense gene protein 1 TP73-AS1 Q9UKY3 Putativeinactive carboxylesterase 4 CES1P1

The Uniprot IDs set forth in Table 2 refer to the human versions thelisted putative proteins and the sequences of each are available fromthe Uniprot database. Sequences of the listed proteins are alsoavailable for various animals, including various mammals and animals ofveterinary or industrial interest. Accordingly, in some embodiments,compositions and methods of the invention provide for the delivery ofone or more mRNAs encoding a protein chosen from mammalian homologs orhomologs from an animal of veterinary or industrial interest of aprotein listed in Table 2; thus, compositions of the invention maycomprise an mRNA encoding a protein chosen from mammalian homologs orhomologs from an animal of veterinary or industrial interest of aprotein listed in Table 2 along with other components set out herein,and methods of the invention may comprise preparing and/or administeringa composition comprising an mRNA encoding a protein chosen frommammalian homologs or homologs from an animal of veterinary orindustrial interest of a protein listed in Table 2 along with othercomponents set out herein. In some embodiments, mammalian homologs arechosen from mouse, rat, hamster, gerbil, horse, pig, cow, llama, alpaca,mink, dog, cat, ferret, sheep, goat, or camel homologs. In someembodiments, the animal of veterinary or industrial interest is chosenfrom the mammals listed above and/or chicken, duck, turkey, salmon,catfish, or tilapia.

In embodiments, the compositions and methods of the invention providefor the delivery of mRNA encoding a lysosomal protein chosen from Table3. In some embodiments, the compositions and methods of the inventionprovide for the delivery of one or more mRNAs encoding one or morelysosomal and/or related proteins listed in Table 3; thus, compositionsof the invention may comprise an mRNA encoding a protein listed in Table3 (or a homolog thereof, as discussed below) along with other componentsset out herein, and methods of the invention may comprise preparingand/or administering a composition comprising an mRNA encoding a proteinchosen from the proteins listed in Table 3 (or a homolog thereof, asdiscussed below) along with other components set out herein.

TABLE 3 Lysosomal and Related Proteins α-fucosidase α-galactosidaseα-glucosidase α-Iduronidase α-mannosidase α-N-acetylgalactosaminidase(α-galactosidase B) β-galactosidase β-glucuronidase β-hexosaminidaseβ-mannosidase 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase3-methylcrotonyl-CoA carboxylase 3-O-sulfogalactosyl cerebrosidesulfatase (arylsulfatase A) acetyl-CoA transferase acidalpha-glucosidase acid ceramidase acid lipase acid phosphatase acidsphingomyelinase alpha-galactosidase A arylsulfatase Abeta-galactosidase beta-glucocerebrosidase beta-hexosaminidasebiotinidase cathepsin A cathepsin K CLN3 CLN5 CLN6 CLN8 CLN9 cystinetransporter (cystinosin) cytosolic protein beta3A subunit of the adaptorprotein-3 complex, AP3 formyl-Glycine generating enzyme (FGE)galactocerebrosidase galactose-1-phosphate uridyltransferase (GALT)galactose 6-sulfate sulfatase (also known asN-acetylgalactosamine-6-sulfatase) glucocerebrosidase glucuronatesulfatase glucuronidase glycoprotein cleaving enzymes glycosaminoglycancleaving enzymes glycosylasparaginase (aspartylglucosaminidase) GM2-APHeparan-alpha-glucosaminide N-acetyltransferase (HGSNAT, TMEM76) Heparansulfatase hexosaminidase A lysosomal proteases methylmalonyl-CoA mutasehyaluronidase Iduronate sulfatase LAMP-2 lysosomal α-mannosidaseLysosomal p40 (C2orf18) Major facilitator superfamily domain containing8 protein (MFSD8 or CLN7) N-acetylgalactosamine 4-sulfatase N-acetylglucosamine 6-sulfatase N-acetyl glucosaminidaseN-acetylglucosamine-1-phosphate transferase NPC1 NPC2 palmitoyl-proteinthioesterase palmitoyl-protein thioesterase (CLN1) Saposin A(Sphingolipid activator protein A) Saposin B (Sphingolipid activatorprotein B) Saposin C (Sphingolipid activator protein C) Saposin D(Sphingolipid activator protein D) sialic acid transporter (sialin)sialidase Sialin sulfatase Transmembrane protein 74 (TMEM74)tripeptidyl-peptidase tripeptidyl-peptidase I (CLN2)UDP-N-acetylglucosamine-phosphotransferase

Information regarding lysosomal proteins is available from Lubke et al.,“Proteomics of the Lysosome,” Biochim Biophys Acta. (2009) 1793:625-635. In some embodiments, the protein listed in Table 3 and encodedby mRNA in the compositions and methods of the invention is a humanprotein. Sequences of the listed proteins are also available for variousanimals, including various mammals and animals of veterinary orindustrial interest. Accordingly, in some embodiments, compositions andmethods of the invention provide for the delivery of one or more mRNAsencoding a protein chosen from mammalian homologs or homologs from ananimal of veterinary or industrial interest of a protein listed in Table3; thus, compositions of the invention may comprise an mRNA encoding aprotein chosen from mammalian homologs or homologs from an animal ofveterinary or industrial interest of a protein listed in Table 3 alongwith other components set out herein, and methods of the invention maycomprise preparing and/or administering a composition comprising an mRNAencoding a protein chosen from mammalian homologs or homologs from ananimal of veterinary or industrial interest of a protein listed in TableS3 along with other components set out herein. In some embodiments,mammalian homologs are chosen from mouse, rat, hamster, gerbil, horse,pig, cow, llama, alpaca, mink, dog, cat, ferret, sheep, goat, or camelhomologs. In some embodiments, the animal of veterinary or industrialinterest is chosen from the mammals listed above and/or chicken, duck,turkey, salmon, catfish, or tilapia.

In some embodiments, the compositions and methods of the inventionprovide for the delivery of mRNA encoding a therapeutic protein (e.g.,cytosolic, transmembrane or secreted) such as those listed in Table 4.In some embodiments, the compositions and methods of the inventionprovide for the delivery of an mRNA encoding a therapeutic proteinuseful in treating a disease or disorder (i.e., indication) listed inTable 4; thus, compositions of the invention may comprise an mRNAencoding a therapeutic protein listed or not listed in Table 4 (or ahomolog thereof, as discussed below) along with other components set outherein for treating a disease or disorder (i.e., indication) listed inTable 4, and methods of the invention may comprise preparing and/oradministering a composition comprising an mRNA encoding a such a protein(or a homolog thereof, as discussed below) along with other componentsset out herein for treatment of a disease or disorder listed in Table 4.

TABLE 4 Exemplary Indications and Related Proteins IndicationTherapeutic Protein 3-Methylcrotonyl-CoA carboxylase deficiencyMethylcrotonoyl-CoA carboxylase 3-Methylglutaconic aciduriaMethylglutaconyl-CoA hydratase Actinic keratosis Acute intermittentporphyria Porphobilinogen deaminase Acute lymphocytic leukemia Acutemyeloid leukemia Addison's disease Adenosine deaminase deficiencyAdenosine deaminase Adrenoleukodystrophy ABCD1 AdrenomyeloneuropathyAIDS/HIV Alcohol use disorders Alkaptonuria Homogentisate1,2-dioxygenase Allergic asthma Anti-IgE mAb Allergies (dermatitis,rhinitis) Alopecia areata Alpers' disease POLG Alpers-Huttenlochersyndrome Alpha 1-antitrypsin deficiency Alpha 1 protease inhibitorAlpha-mannosidosis Alpha-D-mannosidase Alport syndrome Alzheimer'sdisease Amyloid light-chain amyloidosis Amyotrophic lateral sclerosis(ALS) Anemia Erythropoietin Aortic valve stenosis Argininemia ArginaseArgininosuccinic acidemia Argininosuccinate lyase Arrhythmogenic rightventricular dysplasia Autism Autosomal dominant and recessiveprogressive external ophthalmoplegia with mitochondrial DNA deletionsAutosomal recessive polycystic kidney disease ARPKD Bacterial infectionsBasal cell carcinoma Batten disease Battenin + others B-cell chroniclymphocytic leukemia Becker muscular dystrophy DystrophinBeta-thalassemia Beta globin Binge eating disorder Bipolar disorderBladder cancer Blepharospasm, Cervical dystonia, Chronic migraine,Botulinum toxin more Bronchiolitis obliterans Brugada syndrome Buerger'sdisease CACNA1A CACNB4-related Episodic Ataxia Type 2 Cancer anddepression Cancer and sexual dysfunction Cancer in pregnancyCarbamylphosphate synthetase deficiency Carbamylphosphate synthetaseCarcinoma of the gallbladder Cardiomyopathy (diabetic) Cardiomyopathy(hypertrophic) Carnitine uptake defect SLC22A5 Catecholaminergicpolymorphic ventricular tachycardia CDKL5-related Atypical Rett SyndromeCeliac disease Cellulitis Cerebrovascular disease Cervix uteri cancerChronic fatigue syndrome Chronic graft versus host disease Chronicidiopathic urticaria Chronic immune thrombocytopenia ThrombopoietinChronic kidney kisease Chronic liver disease Chronic lymphocyticleukemia Chronic myeloid leukemia Chronic pancreatitis Cirrhosis of theliver Citrullinemia, type I Argininosuccinate synthase Classic RettSyndrome Classical galactosemia Galactose-1-phosphateuridylyltransferase Clostridium difficile associated diarrhea Clottingdisorders COAD/COPD Cocaine addiction COL4A5-related disorders Coldcontact urticaria Contraception, female Coronary artery diseases Corpusuteri cancer Corticobasal degeneration Crigler-Najjar syndromeUDP-glucuronosyltransferase Critical limb ischemia CTNS-relatedcystinosis Cutaneous lupus erythematosus Cutaneous neuroendocrinecarcinoma (Merkel Cell) Cystic fibrosis CFTR Cystic fibrosisDeoxyribonuclease I Cystinosis Cystinosin Cystinuria SLC7A9 Dementia(Lewy body) Depression Diabetic foot infections Diabetic foot ulcerDiabetic peripheral neuropathy Diabetic ulcers Diarrhoeal diseasesDiffuse large B-cell lymphoma DiGeorge syndrome Diverticulitis Drug usedisorders Duchenne muscular dystrophy Dystrophin Dysarthria Dyskinesia(levodopa-induced) Early-onset autosomal dominant Alzheimer's diseaseEczema Ehlers-Danlos syndrome, type 1 EIF2B1 EIF2B2 EIF2B3 EIF2B4EIF2B5-related childhood ataxia with central nervous systemhypomyelination/vanishing white matter Eosinophilic esophagitis EpilepsyErectile dysfunction Erythropoietic protoporphyria FerrochelataseEsophageal carcinoma Essential tremor Fabry disease Alpha galactosidaseFamilial adenomatous polyposis APC Familial chylomicronemia Lipoproteinlipase Familial dysbetalipoproteinemia Apolipoprotein E Familialisolated dilated cardiomyopathy Familial mediterranean fever Pyrin(MEFV) Familial melanoma Female infertility Follicle stimulating hormoneFemale sexual dysfunction Fibromyalgia FMR1-related disorders Fracturehealing Fragile X Premature Ovarian Failure Syndrome Fragile X syndromeFMRP Fragile X-Associated Tremor/Ataxia Syndrome Friedreich's ataxiaFrontotemporal dementia Fryns syndrome Galactocerebrosidase deficienciesGALE deficiency Galactose epimerase GALK deficiency GalactokinaseGALT-related galactosemia Gastric cancer Gastroesophageal reflux diseaseGaucher disease Glucocerebrosidase Gilbert syndromeUDP-glucuronosyltransferase Glioblastoma multiforme GlomerulonephritisGlutaric acidemia, type I Glutaryl-CoA dehydrogenase GM2 gangliosidosisHEXA, HEXB Gout Urate oxidase Graft versus host disease Growth hormonedeficiency Growth hormone 1/Growth hormone 2 Head and neck cancer,Metastatic colorectal cancer Anti-EGFr mAb Hearing loss, adult onsetHeart failure Hemachromatosis HFE protein Hemifacial spasm Hemolyticuremic syndrome Anti-complement factor C5 mAb Hemophilia A Factor VIIIHemophilia A, Hemophilia B Factor VII Hemophilia B Factor IX HepatitisB, Hepatitis C Interferon alpha HER2+ breast cancer, gastric cancerAnti-HER2 mAb Hereditary angioedema C1 esterase inhibitor Hereditaryhemorrhagic telangiectasia Hereditary hemorrhagic telangiectasia (AT)Hereditary spherocytosis Hidradenitis suppurativa HomocystinuriaCystathionine beta-synthase Homozygous familial hypercholesterolemia LDLreceptor Hunter syndrome (MPS II) Iduronate-2-sulfatase Huntingtondisease Huntingtin Hurler syndrome (MPS I) Alpha-L iduronidaseHydrolethalus Hyperalgesia Hyperbilirubinemia HyperhidrosisHyperlipidemia Hypermethioninemia Methionine adenosyltransferaseHyperoxaluria, type I Serine-pyruvate aminotransferase HypertensionHyperuricemia Hyponatremia Hypoparathyroidism Parathyroid hormoneHypophosphatasia TNSALP Idiopathic pulmonary fibrosis IminoglycinuriaImmunoglobulin deficiency Immunoglobulin Infection (adenovirus)Infection (anthrax prophylaxis) Infection (BK virus) Infection(Clostridium difficile prophylaxis) Infection (Dengue fever prophylaxis)Infection (Epstein-Barr virus) Infection (Hepatitis-D) Infection (Lymedisease prophylaxis) Infection (Smallpox virus) Infectious diseasesvaccines Infectious antigen Inflammatory heart diseases InsomniaInterstitial cystitis Iron-deficiency anaemia Irritable bowel diseaseIschaemic heart disease Isovaleric aciduria Isovaleric acid CoAdehydrogenase deficiency Jansky-Bielschowsky disease Juvenile Battendisease Juvenile Neuronal Ceroid Lipofuscinosis (JNCL) Juvenilerheumatoid arthritis TNF-alpha inhibitors Kennedy's disease (SBMA)Keratoconus Krabbe disease Galactocerebrosidase Leber's hereditary opticneuropathy NADH dehydrogenase Leiomyosarcoma Lennox-Gastaut syndromeLesch-Nyhan syndrome Hypoxanthine phosphoribosyltransferase 1 LeukaemiaLi-Fraumeni syndrome TP53 Lipoma Liposarcoma Liver cancer Long-chain3-OH acyl-CoA dehydrogenase deficiency Long-chain-3-hydroxyacyl-CoAdehydrogenase Lower respiratory infections Lysosomal acid lipasedeficiency Lysosomal acid lipase Macular degeneration Major depressivedisorder Malignant fibrous histiocytoma Mantle cell lymphoma Maple syrupurine disease 3-methyl-2-oxobutanoate dehydrogenase Marfan syndrome FBN1Maroteaux-Lamy syndrome (MPS VI) N-acetylgalactosamine 4-sulfataseMastocytosis McArdle disease Muscle glycogen phosphorylase MECP2-relateddisorders MECP2-related Severe Neonatal Encephalopathy Medium-chainacyl-CoA dehydrogenase deficiency Acyl-CoA dehydrogenase MelanomaAnti-CTLA4 mAb Metachromatic leukodystrophy Arylsulfatase A Metastaticcolorectal cancer, NSCLC, others Anti-VEGF mAb Methylmalonyl-CoA mutasedeficiency Methylmalonyl-CoA mutase Migraine Mitochondrial oxidativephosphorylation disorders Morquio syndrome, type A (MPS IVA) Galactose6-sulfate sulfatase Morquio syndrome, type B (MPS IVB)Beta-galactosidase Mouth and oropharynx cancers Multiple carboxylasedeficiency Biotin-methylcrotonoyl-CoA-carboxylase ligase Multiplemyeloma Multiple sclerosis Anti-VLA-4 mAb Multiple sclerosis Interferonbeta Multiple system atrophy Myasthenia gravis Myelofibrosis NarcolepsyNeonatal bronchopulmonary dysplasia Neonatal infections Nephritis andnephrosis Neurofibromatosis, type 1 NF-1 Neuronal ceroidlipofuscinoses-related diseases Neutropenia G-CSF Niemann Pick disease,type A/B SMPD1 Niemann Pick disease, type C NPC1 Niemann-Pick diseaseType C1 Nocturia Non-alcoholic fatty liver disease Non-Hodgkin lymphomaAnti-CD20 mAb Non-small cell lung cancer Notch-3 related cerebralautosomal dominant arteriopathy with subcortical infarcts andleukoencephalopathy (CADASIL) Obesity Ophthalmoparesis Opioid inducedconstipation Ornithine transcarbamylase deficiency Ornithinetranscarbamylase Osteoarthritis Osteopetrosis Osteoporosis Anti-RANKLmAb Ovarian cancer Paget disease of bone Sequestosome 1 Pain Pancreaticcarcinoma Panic disorder Parkinson disease Paroxysmal nocturnalhemoglobinuria Anti-complement factor C5 Mab Pediculosis capitis (headlice) Pelizaeus-Merzbacher disease Pemphigus vulgaris Peptic ulcerdisease Peripheral neuropathy Peyronie's disease PhenylketonuriaPhenylalanine hydroxylase Pneumococcal infection prophylaxisPOLG-related sensory ataxic neuropathy Polycystic kidney diseasePolycystic ovary syndrome Polycythaemia vera Polymerase G-relateddisorders Polymorphous light eruption Pompe disease Alpha glucosidasePorphyria cutanea tarda Uroporphyrinogen decarboxylase Post herpeticneuralgia Post-organ transplant Pouchitis PPM-X Syndrome Prader-Willisyndrome Preeclampsia Premature ejaculation Prematurity and low birthweight Primary ciliary dyskinesia Primary glomerular diseases Primaryhumoral immune deficiencies (e.g., CVID) Immunoglobulin ProctitisProgressive multifocal leukoencephalopathy Progressive supranuclearpalsy Propionic acidemia Propionyl-CoA carboxylase Prostate cancerPsoriasis Anti-IL-12 & IL-23 mAb Psoriatic arthritis TNF-alphainhibitors PTT-1 Pulmonary arterial hypertension Pulmonary arterialhypertension Raynaud's phenomenon Refractive errors Renal cell carcinomaRestless leg syndrome Retinitis pigmentosa Rheumatic heart diseaseRheumatoid arthritis Anti-interleukin-6 (IL-6) mAb Rheumatoid arthritisT-cell costimulation blocker Rheumatoid arthritis TNF-alpha inhibitorRomano-Ward syndrome Rosacea Sanfilippo syndrome, type A (MPS IIIA)Heparan N-sulfatase Sanfilippo syndrome, type B (MPS IIIB)N-acetyl-alpha-D-glucosaminidase Santavuori-Haltia disease SchizophreniaSchnitzler syndrome Scleroderma SCN1A SCN1B-related seizure disordersShort-chain acyl-CoA dehydrogenase deficiency Butyryl-CoA dehydrogenaseSickle cell disease Hemoglobin SLC3A1-related disorders Small cell lungcancer SMN-1-related spinal muscular atrophy (SMA) Spinal muscularatrophy Survival motor neuron protein Squamous cell carcinoma of headand neck Stickler syndrome Stomach cancer Stroke prophylaxis Synovialsarcoma Systemic lupus erythematosus Anti-BAFF Systemic sclerosisTetrahydrobiopterin-deficient hyperphenylalaninemia TetrahydrobiopterinThromboangiitis obliterans Thrombotic disorders Thyroid cancer TPP1deficiencies Trachea, bronchus, lung cancers Tricuspid atresia TSC1TSC2-related tuberous sclerosis Type 2 diabetes mellitus Glucagon-likepeptide 1 (GLP-1) agonist Type 2 diabetes mellitus Insulin Tyrosinemia,type I Fumarylacetoacetase Ulcerative colitis Uterine fibroids Varicoseveins Venous thromboembolism Very long-chain acyl-CoA dehydrogenasedeficiency Long-chain-acyl-CoA dehydrogenase von Gierke's diseaseGlucose-6-phosphatase Von Hippel-Lindau disease pVHL Wegenergranulomatosis Wilson disease Wilson disease protein X-Linked adrenalhypoplasia X-linked adrenoleukodystrophy X-linked agammaglobulinemiaBruton's tyrosine kinase

In some embodiments, one or more therapeutic proteins of the currentinvention are selected from Table 1, 2, 3 or 4. In some specificembodiments, one or more therapeutic proteins are selected from thegroup consisting of alpha galactosidase, erythropoietin, α1-antitrypsin,carboxypeptidase N, alpha-L-iduronidase, iduronate-2-sulfatase,N-acetylglucosamine-1-phosphate transferase, N-acetylglucosaminidase,lysosomal acid lipase, arylsulfatase-A alpha-glucosaminideacetyltransferase, N-acetylglucosamine 6-sulfatase,N-acetylgalactosamine-4-sulfatase, beta-glucosidase, galactose-6-sulfatesulfatase, beta-galactosidase, beta-glucuronidase, glucocerebrosidase,heparan sulfamidase, hyaluronidase, galactocerebrosidase, human growthhormone, ornithine transcarbamylase (OTC), carbamyl phosphatesynthetase-1 (CPS1), argininosuccinate synthetase-1 (ASS1),argininosuccinate lyase (ASL), arginase-1 (ARG1), cystic fibrosistransmembrane conductance regulator (CFTR), Factor VII, Factor VIII,Factor IX, heparan-N-sulfatase, and combinations thereof.

In some embodiments, the present invention is used to prevent, treatand/or cure a subject affected with a disease or disorder listed orassociated with the proteins listed in Tables 1, 2, 3 or 4. Diseases ordisorders for which the compositions and methods of the invention may beemployed include, but are not limited to, disorders such as SMN1-relatedspinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS),GALT-related galactosemia, Cystic Fibrosis (CF), SLC3A1-relateddisorders, cystinuria, COL4A5-related disorders, Alport syndrome,galactocerebrosidase deficiencies, X-linked adrenoleukodystrophy,adrenomyeloneuropathy, Friedreich's ataxia, Pelizaeus-Merzbacherdisease, TSC1 or TSC2-related tuberous sclerosis, Sanfilippo B syndrome(MPS IIIB), CTNS-related cystinosis, the FMR1-related disorders, includeFragile X syndrome, Fragile X-Associated Tremor/Ataxia Syndrome, FragileX Premature Ovarian Failure Syndrome, Prader-Willi syndrome, Fabrydisease, hereditary hemorrhagic telangiectasia (AT), Niemann-Pickdisease Type C1, neuronal ceroid lipofuscinoses-related diseases,Juvenile Neuronal Ceroid Lipofuscinosis (JNCL), Juvenile Batten disease,Santavuori-Haltia disease, Jansky-Bielschowsky disease, PTT-1deficiency, TPP1 deficiency, EIF2B1, EIF2B2, EIF2B3, EIF2B4 andEIF2B5-related childhood ataxia with central nervous systemhypomyelination/vanishing white matter, CACNA1A and CACNB4-relatedEpisodic Ataxia Type 2, the MECP2-related disorders, Classic RettSyndrome, MECP2-related Severe Neonatal Encephalopathy, PPM-X Syndrome,CDKL5-related Atypical Rett Syndrome, Kennedy's disease (SBMA), Notch-3related cerebral autosomal dominant arteriopathy with subcorticalinfarcts and leukoencephalopathy (CADASIL), SCN1A and SCN1B-relatedseizure disorders, Polymerase G-related disorders, Alpers-Huttenlochersyndrome, POLG-related sensory ataxic neuropathy, dysarthria,ophthalmoparesis, autosomal dominant and recessive progressive externalophthalmoplegia with mitochondrial DNA deletions, X-Linked adrenalhypoplasia, X-linked agammaglobulinemia, Wilson's disease, and bloodclotting disorders.

In certain embodiments, the mRNA used in the compositions and methods ofthe invention may encode an antibody. In some embodiments, the mRNA mayencode a protein that is made up of subunits that are encoded by morethan one gene. For example, the protein may be a heterodimer, whereineach chain or subunit of the is encoded by a separate gene.Alternatively, a single mRNA may be engineered to encode more than onesubunit. In one embodiment, the mRNA may encode full length antibodies(both heavy and light chains of the variable and constant regions) orfragments of antibodies (e.g. Fab, Fv, or a single chain Fv (scFv) toconfer immunity to a subject.

As used herein, the term “heavy chain” encompasses all types ofnaturally-occurring heavy chains of different classes ofimmunoglobulins, including but not limited to, IgM(μ), IgD (δ), IgG(γ),IgA(α), and IgE(ε), and biologically active variants thereof. Typically,a heavy chain according to the present invention contains the N-terminalvariable region responsible for antigen recognition, typically includingCDR 1, CDR 2 and CDR 3, separated by four framework regions (FR1, FR2,FR2, and FR4). Typically, the N-terminal variable region contains about100 to 110 or more amino acids. In some embodiments, a heavy chainaccording to the present invention contains one or more of constantdomains (e.g., C_(H)1, C_(H)2, and/or C_(H)3). In some embodiments, anmRNA encoding a heavy chain of an antibody is of or greater than 0.3 kb,0.5 kb, 0.75 kb, 1.0 kb, 1.25 kb, 1.5 kb, 1.75 kb, 2.0 kb, 2.5 kb, 3.0kb, 3.5 kb, 4.0 kb in length.

As used herein, the term “light chain” encompasses all types ofnaturally-occurring light chains of different classes ofimmunoglobulins, including but not limited to κ or λ isotypes, andbiologically active variants thereof. Typically, a light chain accordingto the present invention comprises an N-terminal variable domain(V_(L)). In some embodiments, a light chain according to the presentinvention contains a C-terminal constant domain (C_(L)). In someembodiments, an mRNA encoding a light chain of an antibody is of orgreater than 0.1 kb, 0.2 kb, 0.3 kb, 0.4 kb, 0.5 kb, 0.6 kb, 0.7 kb, 0.8kb, 0.9 kb, 1.0 kb, 1.25 kb, 1.5 kb, 1.75 kb, 2.0 kb, 2.5 kb, or 3.0 kbin length.

According to the present invention, a heavy chain and light chain of anantibody may be encoded and delivered by a single mRNA or separatemRNAs. It is contemplated that it may be advantageous to deliver heavychain encoding mRNA and light chain encoding mRNA at varying ratios inorder to optimize production of fully assembled functional antibodies.

In some embodiments, the mRNA may additionally encode one or moresecretory leader sequences which are operably linked to and directsecretion of an antibody, antibody fragment(s), or other protein(s).Suitable secretory leader sequences are described, for example, in US2008/0286834 A1. While one embodiment of the present invention relatesto methods and compositions useful for conferring immunity to a subject(e.g., via the translation of mRNA encoding functional antibodies), theinventions disclosed herein and contemplated hereby are broadlyapplicable. In an alternative embodiment the compositions of the presentinvention encode antibodies that may be used to transiently orchronically effect a functional response in subjects. For example, themRNA of the present invention may encode a functional monoclonal orpolyclonal antibody, which upon translation and secretion from targetcell may be useful for targeting and/or inactivating a biological target(e.g., a stimulatory cytokine such as tumor necrosis factor).

Lipid Carrier Vehicles

The use of lipid carrier vehicles to facilitate the delivery of nucleicacids to target cells is contemplated by the present invention. Lipidcarrier vehicles (e.g., liposomes and lipid-derived nanoparticles) aregenerally useful in a variety of applications in research, industry, andmedicine, particularly for their use as transfer vehicles of diagnosticor therapeutic compounds in vivo (Lasic, Trends Biotechnol., 16:307-321, 1998; Drummond et al., Pharmacol. Rev., 51: 691-743, 1999) andare usually characterized as microscopic vesicles having an interioraqua space sequestered from an outer medium by a membrane of one or morebilayers. Bilayer membranes of liposomes are typically formed byamphiphilic molecules, such as lipids of synthetic or natural originthat comprise spatially separated hydrophilic and hydrophobic domains(Lasic, Trends Biotechnol., 16: 307-321, 1998). Bilayer membranes of theliposomes can also be formed by amphiphilic polymers and surfactants(e.g., polymerosomes, niosomes, etc.).

In the context of the present invention, a lipid carrier vehicletypically serves to transport mRNA to a target cell. One unexpected andadvantageous feature of the current invention, was the observation thatpulmonary administration of mRNA, which is encapsulated within a lipidcarrier vehicle, results in delivery of mRNA and/or the protein tonon-lung tissue and cells. For the purposes of the present invention,the liposomal transfer vehicles are prepared to contain the desirednucleic acids. The process of incorporation of a desired entity (e.g., anucleic acid) into a liposome is often referred to as “loading” (Lasic,et al., FEBS Lett., 312: 255-258, 1992). The liposome-incorporatednucleic acids may be completely or be partially located in the interiorspace of the liposome, within the bilayer membrane of the liposome, orassociated with the exterior surface of the liposome membrane. Theincorporation of a nucleic acid into liposomes is also referred toherein as “encapsulation” wherein the nucleic acid is entirely containedwithin the interior space of the liposome. The purpose of incorporatinga mRNA into a transfer vehicle, such as a liposome, is often to protectthe nucleic acid from an environment which may contain enzymes orchemicals that degrade nucleic acids and/or systems or receptors thatcause the rapid excretion of the nucleic acids. Accordingly, in someembodiments of the present invention, the selected transfer vehicle iscapable of enhancing the stability of the mRNA contained therein. Theliposome can allow the encapsulated mRNA to reach the target cell and/ormay preferentially allow the encapsulated mRNA to reach non-lung tissueand cells, following pulmonary delivery.

In some embodiments, a suitable lipid carrier vehicle is formulated as alipid nanoparticle. As used herein, the phrase “lipid nanoparticle” and“lipid carrier vehicle” and “lipid-derived nanoparticle” are all usedinterchangeably, and refer to a delivery vehicle comprising one or morelipids (e.g., cationic lipids, non-cationic lipids, cholesterol-basedlipids, and PEG-modified lipids). The contemplated lipid nanoparticlesmay be prepared by including multi-component lipid mixtures of varyingratios employing one or more cationic lipids, non-cationic lipids,cholesterol-based lipids, and PEG-modified lipids. Examples of suitablelipids include, for example, the phosphatidyl compounds (e.g.,phosphatidylglycerol, phosphatidylcholine, phosphatidylserine,phosphatidylethanolamine, sphingolipids, cerebrosides, andgangliosides).

Cationic liposome/mRNA complexes can help to protect mRNA from enzymaticdegradation and facilitate intracellular delivery by interacting withthe negatively charged cell membrane. However, the cationic surface ofthese lipoplexes also mediates strong interactions with negativelycharged proteins that serve to reduce the half-life of the lipoplexes invivo. This effect may be reduced by employing one or more of a mechanismto reduce the interaction between the cationic liposome/mRNA complex andnegatively charged proteins. In most embodiments, the delivery vehiclesused in the compositions and methods of the invention comprisenanoparticles constructed from a combination of one or more cationiclipids, non-cationic lipids, such as neutral or helper lipids, andPEG-modified lipids.

Lipid Nanoparticles

In some embodiments, a suitable delivery vehicle is formulated as alipid nanoparticle. Lipid nanoparticles of the current inventioncomprise one or more lipids (e.g., cationic lipids, non-cationic lipids,cholesterol-based lipids, and PEG-modified lipids). Also contemplated isthe use of polymers as transfer vehicles, whether alone or incombination with other delivery vehicles. In some embodiments, thedelivery vehicle is selected based upon its ability to facilitatepulmonary delivery and translocation to non-lung tissue.

As used herein, liposomal delivery vehicles, e.g. lipid nanoparticles,are usually characterized as microscopic vesicles having an interioraqua space sequestered from an outer medium by a membrane of one or morebilayers. Bilayer membranes of liposomes are typically formed byamphiphilic molecules, such as lipids of synthetic or natural originthat comprise spatially separated hydrophilic and hydrophobic domains(Lasic, Trends Biotechnol., 16: 307-321, 1998). Bilayer membranes of theliposomes can also be formed by amphiphilic polymers and surfactants(e.g., polymerosomes, niosomes, etc.). In the context of the presentinvention, a liposomal delivery vehicle typically serves to transport adesired mRNA to a target tissue. The incorporation of a nucleic acidinto liposomes is also referred to herein as “encapsulation” wherein thenucleic acid is entirely contained within the interior space of theliposome. The purpose of incorporating a mRNA into a transfer vehicle,such as a liposome, is often to protect the nucleic acid from anenvironment which may contain enzymes or chemicals that degrade nucleicacids and/or systems or receptors that cause the rapid excretion of thenucleic acids. Accordingly, in some embodiments, a suitable deliveryvehicle is capable of enhancing the stability of the mRNA containedtherein and/or facilitate the delivery of mRNA to the target cell ortissue.

In certain embodiments of the invention, the carrier is formulated usinga polymer as a carrier, alone or in combination with other carriers.Suitable polymers may include, for example, polyacrylates,polyalkylcyanoacrylates, polylactide, polylactide-polyglycolidecopolymers, polycaprolactones, dextran, albumin, gelatin, alginate,collagen, chitosan, cyclodextrins, protamine, PEGylated protamine, PLL,PEGylated PLL and polyethylenimine (PEI). When PEI is present, it may bebranched PEI of a molecular weight ranging from 10 to 40 kDA, e.g., 25kDa branched PEI (Sigma #408727).

In some embodiments, a suitable delivery vehicle contains a cationiclipid. As used herein, the phrase “cationic lipid” refers to any of anumber of lipid species that have a net positive charge at a selectedpH, such as physiological pH. Several cationic lipids have beendescribed in the literature, many of which are commercially available.Particularly suitable cationic lipids for use in the compositions andmethods of the invention include those described in international patentpublications WO 2010/053572 (and particularly, CI 2-200 described atparagraph [00225]) and WO 2012/170930, both of which are incorporatedherein by reference. In certain embodiments, the compositions andmethods of the invention employ a lipid nanoparticles comprising anionizable cationic lipid described in U.S. provisional patentapplication 61/617,468, filed Mar. 29, 2012 (incorporated herein byreference), such as, e.g,(15Z,18Z)—N,N-dimethyl-6-(9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-15,18-dien-1-amine(HGT5000),(15Z,18Z)—N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-4,15,18-trien-1-amine(HGT5001), and(15Z,18Z)—N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-5,15,18-trien-1-amine(HGT5002).

In some embodiments, the cationic lipidN-[1-(2,3-dioleyloxyl)propyl]-N,N,N-trimethylammonium chloride or“DOTMA” is used. (Feigner et al. (Proc. Nat'l Acad. Sci. 84, 7413(1987); U.S. Pat. No. 4,897,355). DOTMA can be formulated alone or canbe combined with the neutral lipid, dioleoylphosphatidyl-ethanolamine or“DOPE” or other cationic or non-cationic lipids into a liposomaltransfer vehicle or a lipid nanoparticle, and such liposomes can be usedto enhance the delivery of nucleic acids into target cells. Othersuitable cationic lipids include, for example,5-carboxyspermylglycinedioctadecylamide or “DOGS,”2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminiumor “DOSPA” (Behr et al. Proc. Nat.'l Acad. Sci. 86, 6982 (1989); U.S.Pat. No. 5,171,678; U.S. Pat. No. 5,334,761),1,2-Dioleoyl-3-Dimethylammonium-Propane or “DODAP”,1,2-Dioleoyl-3-Trimethylammonium-Propane or “DOTAP”. Contemplatedcationic lipids also include1,2-distearyloxy-N,N-dimethyl-3-aminopropane or “DSDMA”,1,2-dioleyloxy-N,N-dimethyl-3-aminopropane or “DODMA”,1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane or “DLinDMA”,1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane or “DLenDMA”,N-dioleyl-N,N-dimethylammonium chloride or “DODAC”,N,N-distearyl-N,N-dimethylarnrnonium bromide or “DDAB”,N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide or “DMRIE”,3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propaneor “CLinDMA”,2-[5′-(cholest-5-en-3-beta-oxy)-3′-oxapentoxy)-3-dimethyl-1-(cis,cis-9′,1-2′-octadecadienoxy)propaneor “CpLinDMA”, N,N-dimethyl-3,4-dioleyloxybenzylamine or “RE-1”(di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate) or “RE-2”((6Z,25Z)-diethyl16-((4-(dimethylamino)butanoyl)oxy)hentriaconta-6,25-dienedioate) or“RE-3” ((9Z,28Z)-dimethyl19-((4-(dimethylamino)butanoyl)oxy)heptatriaconta-9,28-dienedioate)(See, US2012/0027803, herein incorporated by reference) or “GL-67”(Andries et al., Molecular Pharmaceutics, 9: 2136-2145 (2012); Zhao etal., “Cationic Liposomes in Different Structural Levels for GeneDelivery”, Non-Vrial Gene Therapy, InTech publishing, 13: 293-318(2011), both of which are herein incorporated by reference) or “DMOBA”,1,2-N,N′-dioleylcarbamyl-3-dimethylaminopropane or “DOcarbDAP”,2,3-Dilinoleoyloxy-N,N-dimethylpropylamine or “DLinDAP”,1,2-N,N′-Dilinoleylcarbamyl-3-dimethylaminopropane or “DLincarbDAP”,1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane or “DLinCDAP”,2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane or “DLin-DMA”,2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane or“DLin-K-XTC2-DMA”, and2-(2,2-di((9Z,12Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethanamine(DLin-KC2-DMA)) (See, WO 2010/042877; Semple et al., Nature Biotech. 28:172-176 (2010)), or mixtures thereof (Heyes, J., et al., J ControlledRelease 107: 276-287 (2005); Morrissey, D V., et al., Nat. Biotechnol.23(8): 1003-1007 (2005); PCT Publication WO2005/121348A1). In someembodiments, the cationic lipid is not “GL-67”.

In some embodiments, one or more of the cationic lipids present in sucha composition comprise at least one of an imidazole, dialkylamino, orguanidinium moiety.

In some embodiments, one or more of the cationic lipids present in sucha composition are chosen from XTC(2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane), MC3(((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate), ALNY-100((3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine)),NC98-5(4,7,13-tris(3-oxo-3-(undecylamino)propyl)-N1,N16-diundecyl-4,7,10,13-tetraazahexadecane-1,16-diamide),DODAP (1,2-dioleyl-3-dimethylammonium propane), HGT4003 (WO 2012/170889,the teachings of which are incorporated herein by reference in theirentirety), ICE (WO 2011/068810, the teachings of which are incorporatedherein by reference in their entirety), HGT5000 (U.S. Provisional PatentApplication No. 61/617,468, the teachings of which are incorporatedherein by reference in their entirety) or HGT5001 (cis or trans)(Provisional Patent Application No. 61/617,468), aminoalcohol lipidoidssuch as those disclosed in WO2010/053572, DOTAP(1,2-dioleyl-3-trimethylammonium propane), DOTMA(1,2-di-O-octadecenyl-3-trimethylammonium propane), DLinDMA (Heyes, J.;Palmer, L.; Bremner, K.; MacLachlan, I. “Cationic lipid saturationinfluences intracellular delivery of encapsulated nucleic acids” J.Contr. Rel. 2005, 107, 276-287), DLin-KC2-DMA (Semple, S. C. et al.“Rational Design of Cationic Lipids for siRNA Delivery” Nature Biotech.2010, 28, 172-176), C12-200 (Love, K. T. et al. “Lipid-like materialsfor low-dose in vivo gene silencing” PNAS 2010, 107, 1864-1869).

In some embodiments, a suitable delivery vehicle contains one or morenon-cationic lipids, In some embodiments, a non-cationic lipid is aneutral lipid, i.e., a lipid that does not carry a net charge in theconditions under which the composition is formulated and/oradministered. Such exemplary non-cationic or neutral lipids can bechosen from DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC(1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE(1,2-dioleyl-sn-glycero-3-phosphoethanolamine), DPPE(1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE(1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DOPG(1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol)), and cholesterol.

The use of cholesterol-based cationic lipids is also contemplated by thepresent invention. Such cholesterol-based cationic lipids can be used,either alone or in combination with other cationic or non-cationiclipids. Suitable cholesterol-based cationic lipids include, for example,DC-Choi (N,N-dimethyl-N-ethylcarboxamidocholesterol),1,4-bis(3-N-oleylamino-propyl)piperazine (Gao, et al. Biochem. Biophys.Res. Comm. 179, 280 (1991); Wolf et al. BioTechniques 23, 139 (1997);U.S. Pat. No. 5,744,335), or ICE.

In other embodiments, suitable lipid nanoparticles comprising one ormore cleavable lipids, such as, for example, one or more cationic lipidsor compounds that comprise a cleavable disulfide (S—S) functional group(e.g., HGT4001, HGT4002, HGT4003, HGT4004 and HGT4005), as furtherdescribed in U.S. Provisional Application No. 61/494,745, the entireteachings of which are incorporated herein by reference in theirentirety.

In addition, several reagents are commercially available to enhancetransfection efficacy. Suitable examples include LIPOFECTIN (DOTMA:DOPE)(Invitrogen, Carlsbad, Calif.), LIPOFECTA INE (DOSPA:DOPE) (Invitrogen),LIPOFECTAMINE2000. (Invitrogen), FUGENE, TRANSFECTAM (DOGS), andEFFECTENE.

In some embodiments, the cationic lipid may comprise a molar ratio ofabout 1% to about 90%, about 2% to about 70%, about 5% to about 50%,about 10% to about 40% of the total lipid present in the transfervehicle, or preferably about 20% to about 70% of the total lipid presentin the transfer vehicle.

The use of polyethylene glycol (PEG)-modified phospholipids andderivatized lipids such as derivatized cerarmides (PEG-CER), includingN-Octanoyl-Sphingosine-1-[Succinyl(Methoxy Polyethylene Glycol)-2000](C8 PEG-2000 ceramide) is also contemplated by the present invention,either alone or preferably in combination with other lipids togetherwhich comprise the transfer vehicle (e.g., a lipid nanoparticle).Contemplated PEG-modified lipids include, but is not limited to, apolyethylene glycol chain of up to 5 kDa in length covalently attachedto a lipid with alkyl chain(s) of C6-C20 length. The addition of suchcomponents may prevent complex aggregation and may also provide a meansfor increasing circulation lifetime and increasing the delivery of thelipid-nucleic acid composition to the target cell, (Klibanov et al.(1990) FEBS Letters, 268 (1): 235-237), or they may be selected torapidly exchange out of the formulation in vivo (see U.S. Pat. No.5,885,613).

Particularly useful exchangeable lipids are PEG-ceramides having shorteracyl chains (e.g., C14 or C18). The PEG-modified phospholipid andderivatized lipids of the present invention may comprise a molar ratiofrom about 0% to about 20%, about 0.5% to about 20%, about 1% to about15%, about 4% to about 10%, or about 2% of the total lipid present inthe liposomal transfer vehicle.

The present invention also contemplates the use of non-cationic lipids.As used herein, the phrase “non-cationic lipid” refers to any neutral,zwitterionic or anionic lipid. As used herein, the phrase “anioniclipid” refers to any of a number of lipid species that carry a netnegative charge at a selected H, such as physiological pH. Non-cationiclipids include, but are not limited to, distearoylphosphatidylcholine(DSPC), dioleoylphosphatidylcholine (DOPC),dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol(DOPG), dipalmitoylphosphatidylglycerol (DPPG),dioleoylphosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylethanolamine (POPE),dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or amixture thereof. Such non-cationic lipids may be used alone, but arepreferably used in combination with other excipients, for example,cationic lipids. When used in combination with a cationic lipid, thenon-cationic lipid may comprise a molar ratio of 5% to about 90%, orpreferably about 10% to about 70% of the total lipid present in thetransfer vehicle.

In particular embodiments, a suitable transfer vehicle (e.g., a lipidnanoparticle) is prepared by combining multiple lipid and/or polymercomponents. For example, a transfer vehicle may be prepared usingC12-200, DOPE, chol, DMG-PEG2K at a molar ratio of 40:30:25:5, or DODAP,DOPE, cholesterol, DMG-PEG2K at a molar ratio of 18:56:20:6, or HGT5000,DOPE, chol, DMG-PEG2K at a molar ratio of 40:20:35:5, or HGT5001, DOPE,chol, DMG-PEG2K at a molar ratio of 40:20:35:5. The selection ofcationic lipids, non-cationic lipids and/or PEG-modified lipids whichcomprise the lipid nanoparticle, as well as the relative molar ratio ofsuch lipids to each other, is based upon the characteristics of theselected lipid(s), the nature of the intended target cells, thecharacteristics of the mRNA to be delivered. Additional considerationsinclude, for example, the saturation of the alkyl chain, as well as thesize, charge, pH, pKa, fusogenicity and toxicity of the selectedlipid(s). Thus the molar ratios may be adjusted accordingly. Forexample, in embodiments, the percentage of cationic lipid in the lipidnanoparticle may be greater than 10%, greater than 20%, greater than30%, greater than 40%, greater than 50%, greater than 60%, or greaterthan 70%. The percentage of non-cationic lipid in the lipid nanoparticlemay be greater than 5%, greater than 10%, greater than 20%, greater than30%, or greater than 40%. The percentage of cholesterol in the lipidnanoparticle may be greater than 10%, greater than 20%, greater than30%, or greater than 40%. The percentage of PEG-modified lipid in thelipid nanoparticle may be greater than 1%, greater than 2%, greater than5%, greater than 10%, or greater than 20%.

In certain embodiments, suitable lipid nanoparticles of the inventioncomprise at least one of the following cationic lipids: C12-200,HGT4003, HGT5000, HGT5001, RE-1, RE-2, RE3, GL-67 and ICE. In somespecific embodiments, a suitable lipid nanopartical is formulatedwithout using the cationic lipid GL-67. In some embodiments, suitabletransfer vehicle comprises cholesterol and/or a PEG-modified lipid. Insome embodiments, suitable transfer vehicles comprises DMG-PEG2K. Insome embodiments, suitable transfer vehicle comprises one of thefollowing lipid combinations: C12-200, DOPE, cholesterol, DMG-PEG2K;DODAP, DOPE, cholesterol, DMG-PEG2K; HGT5000, DOPE, cholesterol,DMG-PEG2K; HGT5001, DOPE, cholesterol, DMG-PEG2K; XTC, DSPC,cholesterol, PEG-DMG; MC3, DSPC, cholesterol, PEG-DMG; and ALNY-100,DSPC, cholesterol, DLinKC2-DMA, DODMA, DLinDMA, CLinDMA PEG-DSG.

The lipid carrier vehicles for use in the compositions of the inventioncan be prepared by various techniques which are presently known in theart. Multilamellar vesicles (MLV) may be prepared conventionaltechniques, for example, by depositing a selected lipid on the insidewall of a suitable container or vessel by dissolving the lipid in anappropriate solvent, and then evaporating the solvent to leave a thinfilm on the inside of the vessel or by spray drying. An aqueous phasemay then added to the vessel with a vortexing motion which results inthe formation of MLVs. Uni-lamellar vesicles (ULV) can then be formed byhomogenization, sonication or extrusion of the multi-lamellar vesicles.In addition, unilamellar vesicles can be formed by detergent removaltechniques.

In certain embodiments of this invention, the compositions of thepresent invention comprise a transfer vehicle wherein the mRNA isassociated on both the surface of the transfer vehicle and encapsulatedwithin the same transfer vehicle. For example, during preparation of thecompositions of the present invention, cationic liposomal transfervehicles may associate with the mRNA through electrostatic interactions.For example, during preparation of the compositions of the presentinvention, cationic liposomal transfer vehicles may associate with themRNA through electrostatic interactions.

In some embodiments, the compositions and methods of the inventioncomprise mRNA encapsulated in a lipid carrier vehicle. In someembodiments, the one or more mRNA species may be encapsulated in thesame lipid carrier vehicle. In some embodiments, the one or more mRNAspecies may be encapsulated in different lipid carrier vehicles. In someembodiments, the mRNA is encapsulated in one or more lipid carriervehicles, which differ in their lipid composition, molar ratio of lipidcomponents, size, charge (Zeta potential), targeting ligands and/orcombinations thereof. In some embodiments, the one or more lipid carriervehicles may have a different composition of cationic lipids, neutrallipid, PEG-modified lipid and/or combinations thereof. In someembodiments the one or more lipid carrier vehicles may have a differentmolar ratio of cationic lipid, neutral lipid, cholesterol andPEG-modified lipid used to create the lipid carrier vehicle.

Delivery Methods

The route of delivery used in the methods of the invention allows fornon-invasive, self-administration of the therapeutic compositions of theinvention. The methods of the invention involve intratracheal orpulmonary administration by aerosolization, nebulization, orinstillation of compositions comprising mRNA encoding a therapeuticprotein in a suitable transfection or lipid carrier vehicles asdescribed above.

Although the local cells and tissues of the lung represent a potentialtarget capable of functioning as a biological depot or reservoir forproduction and secretion of the protein encoded by the mRNA, applicantshave discovered that administration of the compositions of the inventionto the lung via aerosolization, nebulization, or instillation results inthe distribution of even non-secreted proteins outside the lung cells.Without wishing to be bound by any particular theory, it is contemplatedthat nanoparticle compositions of the invention pass, through the lungairway-blood barrier, resulting in translation of the intactnanoparticle to non-lung cells and tissues, such as, e.g., the heart,the liver, the spleen, where it results in the production of the encodedprotein in these non-lung tissues. Thus, the utility of the compositionsand methods of the invention extend beyond production of therapeuticprotein in lung cells and tissues of the lung and can be used todelivery to non-lung target cells and/or tissues They are useful in themanagement and treatment of a large number of diseases, and inparticular peripheral diseases which result from both secreted andnon-secreted protein and/or enzyme deficiencies (e.g., one or morelysosomal storage disorders). In certain embodiments, the compositionsof the invention, used in the methods of the invention result in thedistribution of the mRNA encapsulated nanoparticles and production ofthe encoded protein in the liver, spleen, heart, and/or other non-lungcells. For example, administration of the compositions of the presentinventions, such as, e.g., a nanoparticle comprising mRNA encoding betagalactosidase (a non-secreted protein), by aerosolization, nebulization,or instillation to the lung will result in the composition itself andits protein product (e.g., functional beta galactosidase protein) willbe detectable in both the local cells and tissues of the lung, as wellas in peripheral target cells, tissues and organs as a result oftranslocation of the mRNA and delivery vehicle to non-lung cells.

In certain embodiments, the compositions of the invention may beemployed in the methods of the invention to specifically targetperipheral cells or tissues. Following the pulmonary delivery, it iscontemplated that the compositions of the invention cross the lungairway-blood barrier and distribute into cells other than the local lungcells. Accordingly, the compositions disclosed herein may beadministered to a subject by way of the pulmonary route ofadministration, using a variety of approach known by those skilled inthe art (e.g., by inhalation), and distribute to both the local targetcells and tissues of the lung, as well as in peripheral non-lung cellsand tissues (e.g., cells of the liver, spleen, kidneys, heart, skeletalmuscle, lymph nodes, brain, cerebrospinal fluid, and plasma). As aresult, both the local cells of the lung and the peripheral non-lungcells can serve as biological reservoirs or depots capable of producingand/or secreting a translation product encoded by one or morepolynucleotides. Accordingly, the present inventions are not limited tothe treatment of lung diseases or conditions, but rather can be used asa non-invasive means of facilitating the delivery of polynucleotides, orthe production of enzymes and proteins encoded thereby, in peripheralorgans, tissues and cells (e.g., hepatocytes) which would otherwise beachieved only by systemic administration. Exemplary peripheral non-lungcells include, but are not limited to, hepatocytes, epithelial cells,hematopoietic cells, epithelial cells, endothelial cells, bone cells,stem cells, mesenchymal cells, neural cells, cardiac cells, adipocytes,vascular smooth muscle cells, cardiomyocytes, skeletal muscle cells,beta cells, pituitary cells, synovial lining cells, ovarian cells,testicular cells, fibroblasts, B cells, T cells, reticulocytes,leukocytes, granulocytes and tumor cells.

Following administration of the composition to the subject, the proteinproduct encoded by the mRNA (e.g., a functional protein or enzyme) isdetectable in the peripheral target tissues for at least about one toseven days or longer following administration of the composition to thesubject. The amount of protein product necessary to achieve atherapeutic effect will vary depending on the condition being treated,the protein encoded, and the condition of the patient. For example, theprotein product may be detectable in the peripheral target tissues at aconcentration (e.g., a therapeutic concentration) of at least 0.025-1.5μg/ml (e.g., at least 0.050 μg/ml, at least 0.075 μg/ml, at least 0.1μg/ml, at least 0.2 μg/ml, at least 0.3 μg/ml, at least 0.4 μg/ml, atleast 0.5 μg/ml, at least 0.6 μg/ml, at least 0.7 μg/ml, at least 0.8μg/ml, at least 0.9 μg/ml, at least 1.0 μg/ml, at least 1.1 μg/ml, atleast 1.2 μg/ml, at least 1.3 μg/ml, at least 1.4 μg/ml, or at least 1.5μg/ml), for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35,40, 45 days or longer following administration of the composition to thesubject.

It has been demonstrated that nucleic acids can be delivered to thelungs by intratracheal administration of a liquid suspension of thenucleic acid composition and inhalation of an aerosol mist produced by aliquid nebulizer or the use of a dry powder apparatus such as thatdescribed in U.S. Pat. No. 5,780,014, incorporated herein by reference.

In certain embodiments, the compositions of the invention may beformulated such that they may be aerosolized or otherwise delivered as aparticulate liquid or solid prior to or upon administration to thesubject. Such compositions may be administered with the assistance ofone or more suitable devices for administering such solid or liquidparticulate compositions (such as, e.g., an aerosolized aqueous solutionor suspension) to generate particles that are easily respirable orinhalable by the subject. In some embodiments, such devices (e.g., ametered dose inhaler, jet-nebulizer, ultrasonic nebulizer,dry-powder-inhalers, propellant-based inhaler or an insufflator)facilitate the administration of a predetermined mass, volume or dose ofthe compositions (e.g., about 0.5 mg/kg of mRNA per dose) to thesubject. For example, in certain embodiments, the compositions of theinvention are administered to a subject using a metered dose inhalercontaining a suspension or solution comprising the composition and asuitable propellant. In certain embodiments, the compositions of theinvention may be formulated as a particulate powder (e.g., respirabledry particles) intended for inhalation. In certain embodiments,compositions of the invention formulated as respirable particles areappropriately sized such that they may be respirable by the subject ordelivered using a suitable device (e.g., a mean D50 or D90 particle sizeless than about 500 μm, 400 μm, 300 μm, 250 μm, 200 μm, 150 μm, 100 μm,75 μm, 50 μm, 25 μm, 20 μm, 15 μm, 12.5 μm, 10 μm, 5 μm, 2.5 μm orsmaller). In yet other embodiments, the compositions of the inventionare formulated to include one or more pulmonary surfactants (e.g.,lamellar bodies). In some embodiments, the compositions of the inventionare administered to a subject such that a concentration of at least 0.05mg/kg, at least 0.1 mg/kg, at least 0.5 mg/kg, at least 1.0 mg/kg, atleast 2.0 mg/kg, at least 3.0 mg/kg, at least 4.0 mg/kg, at least 5.0mg/kg, at least 6.0 mg/kg, at least 7.0 mg/kg, at least 8.0 mg/kg, atleast 9.0 mg/kg, at least 10 mg/kg, at least 15 mg/kg, at least 20mg/kg, at least 25 mg/kg, at least 30 mg/kg, at least 35 mg/kg, at least40 mg/kg, at least 45 mg/kg, at least 50 mg/kg, at least 55 mg/kg, atleast 60 mg/kg, at least 65 mg/kg, at least 70 mg/kg, at least 75 mg/kg,at least 80 mg/kg, at least 85 mg/kg, at least 90 mg/kg, at least 95mg/kg, or at least 100 mg/kg body weight is administered in a singledose. In some embodiments, the compositions of the invention areadministered to a subject such that a total amount of at least 0.1 mg,at least 0.5 mg, at least 1.0 mg, at least 2.0 mg, at least 3.0 mg, atleast 4.0 mg, at least 5.0 mg, at least 6.0 mg, at least 7.0 mg, atleast 8.0 mg, at least 9.0 mg, at least 10 mg, at least 15 mg, at least20 mg, at least 25 mg, at least 30 mg, at least 35 mg, at least 40 mg,at least 45 mg, at least 50 mg, at least 55 mg, at least 60 mg, at least65 mg, at least 70 mg, at least 75 mg, at least 80 mg, at least 85 mg,at least 90 mg, at least 95 mg or at least 100 mg mRNA is administeredin one or more doses.

EXAMPLES Example 1 Comparison of Unmodified and Modified mRNA in NakedForm or in Nanoparticles by Intratracheal Administration

Overview: Mice were intratracheally (IT) sprayed either with unmodifiedor modified mRNA (25% of both cytidine-5′-triphosphate anduridine-5′-triphosphate were replaced by5-methylcytidine-5′-triphosphate and 2-thiouridine-5′-triphosphate,respectively) coding for firefly luciferase (FFL) either in naked formor encapsulated in lipid based nanoparticles (NPs) for single doseadministration. Luciferase production was measured by in vivoBioluminescence Imaging (BLI) at different time points post IT spray.Organs from mice treated with C12-200 based NPs at a dose amounting to20 μg/mouse were prepared for histopathological analysis. To assessbiodistribution of complexes post IT spray, in vitro luciferaseproduction was measured in the organs prepared from the euthanized micetreated with doses corresponding to 5 and 10 μg C12-200 based NPs permouse.

A. IT Spray of Naked mRNA and mRNA in C12-200 Based Nanoparticles—20 ugPer Mouse

Lipid Nanoparticle Formulation: Aliquots of 50 mg/mL ethanolic solutionsof C12-200, DOPE, Choleterol, and DMG-PEG2000 were mixed in a molarratio of 40:30:25:5, respectively, and diluted with ethanol to 3 mLfinal volume. Separately, an aqueous buffered solution (10 mMcitrate/150 mM NaCl, pH 4.5) of FFL or modified FFL mRNA was preparedfrom a 1 mg/mL stock. The lipid solution was injected rapidly into theaqueous mRNA solution and shaken to yield a final suspension in 20%ethanol. The resulting nanoparticle suspension was filtered,diafiltrated with 1×PBS (pH 7.4), concentrated and stored at 2-8° C.

Messenger RNA was synthesized via in vitro transcription process using acDNA template encoding FFL protein with predetermined untranslatedregions. The resulting RNA construct was processed further incorporatinga Cap 1 structure on the 5′ end and a poly-A tail length of ˜200adenosine bases.

Modified messenger RNA was synthesized in a similar fashion as statedabove with 25% of the uridine bases substituted with 2-thiouridinetriphosphate and 25% of the cytidine bases substituted with5-methylcytidine triphosphate.

Female Balb/c mice were purchased from Elevage-Janvier, France. The micewere 10 weeks old at the start of the experiment. Mice were weighedprior to the start of the experiment and assigned to one of thefollowing four group (n=6 mice per group): group I—IT spray with FFLmRNA; group II—IT spray with modified FFL mRNA; group III—IT spray withFFL-mRNA in C12-200 based lipid nanoparticle; group IV—IT spray withmodified FFL-mRNA in C12-200 based lipid nanoparticle. Each mouse wassprayed with 20 μg of the respective mRNA/NPs. The required amount ofmRNA/NPs per group were suspended just before application in DEPCtreated (0.1%) RNase free water (Serva, Catalog number: 39798, LotP060383), to a total volume of 50 μl/mouse. NPs were also characterizedby size and zeta potential measurements. These measurements wereperformed in water and are tabulated as Table 5.

TABLE 5 Particle size and zeta potential measurements made in PBS Size(nm) ± polydispersity Zeta Potential FFL-NP 81.64 ± 0.177   +50 ± 6.36FFL-mod-NP 84.14 ± 0.197 +52.5 ± 16.2

Luciferase production was measured by in vivo BLI at 6 hours postapplication. Whereas almost negligible amount of exogenous mRNA-derivedprotein could be detected with naked mRNA, the nanoparticleformulations, independent of modifications, showed significant levels ofluciferase production in the entire thoracic region and upper abdomen(FIG. 1). Compared to modified FFL, the unmodified FFL mRNA resulted inapproximately 2-3 fold higher luminescence at 6 hours (FIG. 2).

(FIGS. 1A and B), very low (close to background) level luminescencecould be detected with naked mRNA. In mice treated with lipidnanoparticles, independent of modifications, increased luciferaseproduction (˜2-3 fold), compared to 6 hour time point, was observed 24hours after treatment (FIGS. 3A and B). No significant difference couldbe observed between luciferase production from FFL or modified FFL mRNA(FIG. 3C).

The mice treated with naked mRNA, were followed further in theexperiment and two additional doses at weekly intervals were applied.BLI was performed at different time points post application. The BLIimages at 24 hours post application, the time point of maximumluminescence (FIG. 3), are shown in FIG. 4 (naked FFL mRNA) and FIG. 5(naked modified FFL mRNA). With a few exceptions, no noticeableluciferase production was observed for any of the measured mice (comparethe scales in FIGS. 4 and 5 with FIG. 3).

B. IT Spray of FFL and Modified FFL mRNA in C12-200 BasedNanoparticles—5 μg Per Mouse and 10 μg Per Mouse

IT spray experiments were performed with reduced doses of 5 and 10μg/mouse. The C12-200 based nanoparticle formulation was as described inExample 1.

Experimental Design: Female Balb/c mice were purchased fromElevage-Janvier, France. The mice were 19 weeks old at the start of theexperiment. Mice were weighed prior to the start of the experiment. TheC12-200 based lipid nanoparticles were suspended just before applicationin DEPC treated (0.1%) RNase free water (Serva, Catalog number: 39798,Lot P060383), to a total volume of 50 μl/mouse. The following fourgroups were tested (n=5 mice per group): group I—IT spray with FFL mRNAin C12-200 based nanoparticles (5 μg/mouse); group II—IT spray with FFLmRNA in C12-200 based nanoparticles (10 μg/mouse); group III—IT spraywith modified FFL mRNA in C12-200 based nanoparticles (5 μg/mouse); andgroup IV—IT spray with modified FFL mRNA in C12-200 based nanoparticles(10 μg/mouse). One mouse from group III and IV died during IT spray.Thus, the remaining number of animals for these groups was four. At 6hours post application, all animals showed piloerection and reducedmotility. Moreover, one mouse from each of the higher dose groups(groups II and IV) was dead at this time point. BLI imaging wasperformed for the mice at 6 hours post application.

Using FFL mRNA at the doses of 5 μg/mouse resulted in extremely lowlevels of luciferase production. With the 10 μg/mouse dose, greaterproduction was observed which was concentrated in the liver (FIG. 6A).The difference between the applied doses was not very evident in themodified FFL mRNA groups (FIG. 6B). Comparing the mice from group II(FFL mRNA) with group IV (modified FFL mRNA) revealed higherluminescence in the former (compare FIG. 6A panel 1, 2 with panel 1 inFIG. 6B).

Luciferase production at 24 hours was significantly enhanced compared to6 hours post IT spray (FIGS. 7A and 7B). Moreover, higher production wasobserved using FFL mRNA as compared to modified FFL mRNA (FIG. 7C).Similar results were obtained in Example 1 with the dose of 20 μg/mouse.Internal organs (heart, liver, lungs, liver, spleen and kidney) werefrozen in liquid nitrogen for in-vitro luciferase measurements.

Biodistribution after IT spray: The isolated organs were homogenized inthe frozen state using a mortar and pestel, weighed and lysed in asolution containing Lysis-buffer (25 mM TRIS-C1 0.1% Triton x-100; pH7.4) and Complete-Protease-Inhibitor (Roche). Spleen, heart and kidneyswere lysed in 250 μl, whereas lungs and liver were lysed in 400 μl.After incubation on ice for 20 min, samples were centrifuged at 10.000rpm, 4° C. for 10 min. Luciferase activity was measured using 100 μl ofthe supernatant. Each sample was measured in duplicates and mean valuesfrom duplicates were used in analysis. All organs except the kidneyswere positive for luciferase activity (FIG. 8). In accordance with ourBLI data, maximum luminescence was observed in liver and lungs. FFL-mRNAresulted in higher protein production compared to modified-FFL-mRNA anda dose dependency was evident.

C. IT Spray of Modified FFL mRNA in HGT5001 Based Nanoparticles—20 μgPer Mouse

An IT spray experiment was performed with an HGT5001 based nanoparticleformulation.

Lipid nanoparticle formulation: Aliquots of 50 mg/mL ethanolic solutionsof HGT5001:DOPE:Cholesterol:DMG-PEG2K were mixed in a molar ratio of40:20:35:5, respectively, and diluted with ethanol to 3 mL final volume.Separately, an aqueous buffered solution (10 mM citrate/150 mM NaCl, pH4.5) of FFL or modified FFL mRNA was prepared from a 1 mg/mL stock. Thelipid solution was injected rapidly into the aqueous mRNA solution andshaken to yield a final suspension in 20% ethanol. The resultingnanoparticle suspension was filtered, diafiltrated with 1×PBS (pH 7.4)followed by distilled RNAse-free water, concentrated and stored at 2-8°C.

Experimental Design: Female Balb/c mice were purchased fromElevage-Janvier, France. The mice were 13 weeks old at the start of theexperiment. Mice were weighed prior to the start of the experiment. Thelipid nanoparticles were suspended just before application in DEPCtreated (0.1%) RNase free water (Serva, Catalog number: 39798, LotP060383), to a total volume of 50 μl/mouse.

IT Spray and BLI: Each mouse was IT sprayed with 20 μg of the HGT5001based nanoparticle formulation in a total volume of 50 μl/mouse. BLIimaging was performed for the mice at 6 hrs post application.

Significantly lower luminescence values were observed with the HGT5001based nanoparticles when compared to the corresponding time point withthe C12-200 based nanoparticles and no increase in protein productionfrom 6 to 24 hours was observed (FIG. 9 and FIG. 10).

In an independent experiment testing C12-200 and HGT5001 basednanoparticle formulations, following BLI imaging (FIG. 11), mice wereeuthanized and organs (heart, lungs, liver, spleen and kidney) wereevaluated by histology (FIG. 12). FFL production was confirmed in thelung and liver for both C12-200 based NPs and HGT5011 based NPs.

D. IT Spray of Modified FFL mRNA—Non-Nanoparticle Delivery

Naked mRNA resulted in low efficiency without perfluorocarbon treatment.IT aerosolization of encapsulated mRNA lead to protein production inlungs, liver, spleen, and heart. FFL and modified FFL were equallyefficient with respect to protein production and with a dose-response.

Various delivery vehicles were tested, including polethylenimines (L-PEI22 kDa, br-PEI 25 kDa), copolymers of oligo(ethylene glycol) methylether methacrylate (OEGMA) and N,N-dimethylaminoethyl methacrylate(DMAEMA), MLRI:DOPE, DOTAP, DMRIE-C, and Lipofectamine, and did not showluminescence in non-lung cells. In contrast, C12-200 and HGT5001 basedlipid nanoparticle formulations resulted in significant proteinproduction in non-lung cells following pulmonary delivery.

These observations indicate that only the nanoparticle formulations wereable to translocate intact, by either active or passive means, from thelung to the systemic blood supply and subsequently to be deposited indifferent tissues, such as the liver. This translocation of an intactmRNA encoding a cytosolic protein, firefly luciferase, constitutesnon-invasive systemic delivery of an active pharmaceutical ingredientbeyond the lung to result in the production of a functional protein tosystemically accessible tissues.

E. Nebulization of Modified FFL mRNA with PEI Based Lipid Nanoparticles

Mice that received modified FFL mRNA in PEI based nanoparticles showedluminescence in the lung (FIG. 13). Luciferase production was greaterwith modified FFL mRNA (panels 1, 2) compared to unmodified FFL mRNA(panels 3, 4).

Example 2 Evaluation of Nanoparticle Migration by Measuring Lipid inNon-Lung Target Cells

To identify the passage of intact nanoparticles in non-lung tissues,aliquots of 50 mg/mL ethanolic solutions of C12-200, DOPE, Chol,DMG-PEG2000 and1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamineB sulfonyl) (ammonium salt) are mixed in a molar ratio of 40:29:25:5:1,respectively, and diluted with ethanol to 3 mL final volume. Separately,an aqueous buffered solution (10 mM citrate/150 mM NaCl, pH 4.5) ofnon-secreted protein, such as beta-galactosidase or FFL (modified orunmodified) mRNA is prepared from a 1 mg/mL stock. The lipid solution isinjected rapidly into the aqueous mRNA solution and shaken to yield afinal suspension in 20% ethanol. The resulting nanoparticle suspensionis filtered, diafiltrated with 1×PBS (pH 7.4), concentrated and storedat 2-8° C.

Messenger RNA is synthesized via in vitro transcription process using acDNA template encoding beta-galactosidase or FFL protein withpredetermined untranslated regions. The resulting mRNA construct isprocessed further incorporating a Cap 1 structure on the 5′ end and apoly-A tail length of ˜200 adenosine bases.

Modified messenger RNA is synthesized in a similar fashion as statedabove with 25% of the uridine bases substituted with 2-thiouridinetriphosphate and 25% of the cytidine bases substituted with5-methylcytidine triphosphate.

Female Balb/c mice are purchased from Elevage-Janvier, France. The miceare 10 weeks old at the start of the experiment. Mice are weighed priorto the start of the experiment. Each mouse is sprayed with 20 μg of therespective mRNA/NPs comprising unmodified and modified mRNA influorescently labeled C12-200 based lipid nanoparticles. The mRNAencapsulated nanoparticles are suspended just before application in DEPCtreated (0.1%) RNase free water (Serva, Catalog number: 39798, LotP060383), to a total volume of 50 μl/mouse. Six hours after treatmentmice are killed and organs are excised for histological examination ofNP distribution by fluorescence microscopy on 6 μm cryosections.

Alternatively, mRNA is radioactively labeled with, e.g. I¹²³ accordingto the method of Commerford as described in detail by Terebesi et al(Terebesi J, Kwok K Y, Rice K G. Anal Biochem. 1998 Oct. 1;263(1):120-3). The labeling mixture is separated using a PD-10 gelfiltration column (Amersham Biosciences, Freiburg, Germany) with wateras eluent. The iodinated mRNA is mixed with unlabeled mRNA resulting inthe desired amounts of mRNA which is formulated with lipids as describedabove and IT aerosolized to the mice lungs. At a desired time point,mice are killed and radioactivity of the organs is measured using agamma counter.

The above examples demonstrate mRNA can be effectively delivered tonon-lung cells or tissues through pulmonary administration using themethods and compositions described herein. In the representativeexamples above, mRNA delivery was evaluated using the fluorescentfirefly luciferase reporter protein encoded by a codon optimizedsequence of modified mRNA. However, it will be appreciated by thoseskilled in the art, that such examples are merely representative of awide range of mRNAs and proteins that can be delivered according to thepresent invention. In particular, it will be readily apparent to oneskilled in the art that the compositions and methods of the currentinvention may be used to delivery mRNA encoding various therapeuticproteins to non-lung cells or tissues within a subject for the treatmentof associated diseases, disorders or conditions.

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

All references, patents or applications, U.S. or foreign, cited in theapplication are hereby incorporated by reference as if written herein intheir entireties. Where any inconsistencies arise, material literallydisclosed herein controls.

1. A method of delivery of messenger RNA (mRNA) to a non-lung cell ortissue comprising administering to the lung a composition comprisingmRNA encoding a protein and a lipid carrier vehicle, wherein theadministering to the lung results in the delivery of the mRNA and/or theprotein to a non-lung cell or tissue.
 2. The method of claim 1, whereinthe composition is administered to the lung by aerosolization,inhalation, nebulization, instillation or intratracheal aerosolization.3. (canceled)
 4. The method of claim 1, wherein the non-lung cell isselected from the group consisting of hepatocytes, epithelial cells,hematopoietic cells, epithelial cells, endothelial cells, bone cells,stem cells, mesenchymal cells, neural cells, cardiac cells, adipocytes,vascular smooth muscle cells, cardiomyocytes, skeletal muscle cells,beta cells, pituitary cells, synovial lining cells, ovarian cells,testicular cells, fibroblasts, B cells, T cells, reticulocytes,leukocytes, granulocytes, macrophages, neutrophils, antigen presentingcells (dendritic cells), fibroblasts, tumor cells and combinationthereof.
 5. (canceled)
 6. The method of claim 1, wherein the non-lungtissue is selected from the group consisting of heart, liver, spleen,kidneys, skeletal muscle, lymph nodes, skin, brain, cerebrospinal fluid,plasma and combination thereof. 7-9. (canceled)
 10. The method of claim1, wherein the mRNA encodes a protein selected from the group consistingof a cytosolic protein, a secreted protein and a therapeutic protein.11. The method of claim 10, wherein the cytosolic protein is selectedfrom the group consisting of enzymes, transcription factors, chaperones,and combination thereof. 12-13. (canceled)
 14. The method of claim 10,wherein the therapeutic protein is selected from the group consisting ofalpha galactosidase, erythropoietin, α1-antitrypsin, carboxypeptidase N,alpha-L-iduronidase, iduronate-2-sulfatase,N-acetylglucosamine-1-phosphate transferase, N-acetylglucosaminidase,lysosomal acid lipase, arylsulfatase-A alpha-glucosaminideacetyltransferase, N-acetylglucosamine 6-sulfatase,N-acetylgalactosamine-4-sulfatase, beta-glucosidase, galactose-6-sulfatesulfatase, beta-galactosidase, beta-glucuronidase, glucocerebrosidase,heparan sulfamidase, hyaluronidase, galactocerebrosidase, human growthhormone, ornithine transcarbamylase (OTC), carbamyl phosphatesynthetase-1 (CPS1), argininosuccinate synthetase-1 (ASS1),argininosuccinate lyase (ASL), arginase-1 (ARG1), cystic fibrosistransmembrane conductance regulator (CFTR), Factor VII, Factor VIII,Factor IX, heparan-N-sulfatase, and combination thereof.
 15. The methodof claim 1, wherein the protein is detectable in the non-lung cell ortissue for at least about 1 day or for at least about 2 days followingthe administration to the lung.
 16. (canceled)
 17. The method of claim1, wherein the mRNA is delivered at an amount greater than about 0.5mg/kg of mRNA per dose.
 18. The method of claim 1, wherein the lipidcarrier vehicle is a liposome.
 19. The method of claim 18, wherein theliposome comprises one or more cationic lipids, one or more non-cationiclipids, one or more cholesterol-based lipids and one or morePEG-modified lipids.
 20. The method of claim 19, wherein the one or morecationic lipids are selected from the group consisting of C12-200,HGT4003, HGT5000, HGT5001, ICE, DLinKC2-DMA, DODAP, DODMA, DLinDMA,CLinDMA, and combination thereof.
 21. The method of claim 1, wherein thecomposition further comprises one or more pulmonary surfactants.
 22. Themethod of claim 1, wherein the composition is formulated as respirableparticles.
 23. The method of claim 22, wherein the respirable particleshave a size less than about 500 μm.
 24. The method of claim 1, whereinthe composition is formulated as nebulizable lipid or as dry powder.25-27. (canceled)
 28. A method of treating a disease or disordercomprising administering to the lung a composition comprising mRNAencoding a therapeutic protein and a lipid carrier vehicle, wherein theadministration to the lung results in the delivery of the therapeuticprotein to a non-lung cell or tissue affected by the disease ordisorder.
 29. The method of claim 28, wherein the disease or disorder isselected from Table
 4. 30. A composition for pulmonary delivery ofmessenger RNA (mRNA) comprising mRNA encoding a protein and a lipidcarrier vehicle, wherein the composition is formulated such that onceadministered to the lung, it results in the delivery of the mRNA and/orthe protein to a non-lung cell or tissue. 31-37. (canceled)
 38. Thecomposition of claim 30, wherein the composition is formulated asnebulizable lipid or as dry power for inhalation. 39-44. (canceled)